Synergistic treatment of cancer using immunomers in conjunction with chemotherapeutic agents

ABSTRACT

The invention relates to the therapeutic use of immunostimulatory oligonucleotides and/or immunomers in combination with chemotherapeutic agents to provide a synergistic therapeutic effect.

RELATED APPLICATION

This application is a Continuation application of U.S. patentapplication Ser. No. 10/846,167 filed May 14, 2004 and claims thebenefit of U.S. Provisional Application No. 60/471,247, filed May 16,2003, which is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to anti-cancer applications using immunomers astherapeutic agents.

2. Summary of the Related Art

Recently, several researchers have demonstrated the validity of the useof oligonucleotides as immunostimulatory agents in immunotherapyapplications. The observation that phosphodiester and phosphorothioateoligonucleotides can induce immune stimulation has created interest indeveloping these compounds as a therapeutic tool. These efforts havefocused on phosphorothioate oligonucleotides containing the naturaldinucleotide CpG. Kuramoto et al., Jpn. J. Cancer Res. 83:1128-1131(1992) teaches that phosphodiester oligonucleotides containing apalindrome that includes a CpG dinucleotide can induce interferon-alphaand gamma synthesis and enhance natural killer activity. Krieg et al.,Nature 371:546-549 (1995) discloses that phosphorothioate CpG-containingoligonucleotides are immunostimulatory. Liang et al., J. Clin. Invest.98:1119-1129 (1996) discloses that such oligonucleotides activate humanB cells. Moldoveanu et al., Vaccine 16:1216-124 (1998) teaches thatCpG-containing phosphorothioate oligonucleotides enhance immune responseagainst influenza virus. McCluskie and Davis, J. Immunol. 161:4463-4466(1998) teaches that CpG-containing oligonucleotides act as potentadjuvants, enhancing immune response against hepatitis B surfaceantigen.

Other modifications of CpG-containing phosphorothioate oligonucleotidescan also affect their ability to act as modulators of immune response.See, e.g., Zhao et al., Biochem. Pharmacol. (1996) 51:173-182; Zhao etal., Biochem Pharmacol. (1996) 52:1537-1544; Zhao et al., AntisenseNucleic Acid Drug Dev. (1997) 7:495-502; Zhao et al., Bioorg. Med. Chem.Lett. (1999) 9:3453-3458; Zhao et al., Bioorg. Med. Chem. Lett. (2000)10:1051-1054; Yu et al., Bioorg. Med. Chem. Lett. (2000) 10:2585-2588;Yu et al., Bioorg. Med. Chem. Lett. (2001) 11:2263-2267; and Kandimallaet al., Bioorg. Med. Chem. (2001) 9:807-813. U.S. Pat. No. 6,426,334shows the promise of these compounds as anti-cancer agents.

Although it has been well demonstrated that many murine and human tumorscarry immunogenic epitopes that can be recognized by the host immunesystem, in most cases host defenses fail to mount an appropriateresponse causing uncontrolled tumor growth in cancer patients. Thefailure of the host immune system to elicit defense against tumor cellscould be related to low immunogenic nature of tumor antigens and/ordefects in the host immune system itself.

These reports make clear that there remains a need to be able to enhancethe anticancer activity of immunostimulatory oligonucleotides.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods for enhancing the anti-cancer activity ofimmunostimulatory oligonucleotide compounds. The methods according tothe invention enable synergy between the immunostimulatory effect ofimmunostimulatory oligonucleotides and the therapeutic effect ofchemotherapeutic agents. Modification of an immunostimulatoryoligonucleotide to optimally present 5′ ends dramatically enhances itsanti-cancer activity. Such an oligonucleotide is referred to herein asan “immunomer”, which may contain one or more immunostimulatoryoligonucleotide.

In a first aspect, therefore, the invention provides methods fortreating cancer in a cancer patient comprising administering to thepatient an immunostimulatory oligonucleotide and/or immunomer incombination with a chemotherapeutic agent, wherein the immunostimulatoryoligonucleotide and/or immunomer and the chemotherapeutic agent create asynergistic therapeutic effect.

In some embodiments, the immunostimulatory oligonucleotide and/orimmunomer used in the method according to the invention comprises animmunostimulatory dinucleotide selected from the group consisting ofCpG, C*pG, CpG*, and C*pG*, wherein C is cytidine or 2′-deoxycytidine,C* is 2′-deoxythymidine.arabinocytidine, 2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substitutedarabinocytidine,2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine,2′-deoxy-4-thiouridine, other non-natural pyrimidine nucleosides, or1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; G isguanosine or 2′-deoxyguanosine, G* is 2′ deoxy-7-deazaguanosine,2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2′substituted-arabinoguanosine, 2′-O-substituted-arabinoguanosine, orother non-natural purine nucleoside, and p is an internucleoside linkageselected from the group consisting of phosphodiester, phosphorothioate,and phosphorodithioate. In certain preferred embodiments, theimmunostimulatory dinucleotide is not CpG.

In some embodiments, the immunostimulatory oligonucleotide and/orimmunomer used in the method according to the invention comprises animmunostimulatory domain of formula (III):

5′-Nn-N1-Y—Z—N1-Nn-3′  (III)

wherein:

Y is cytidine, 2′-deoxythymidine, 2′-deoxycytidine, arabinocytidine,2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substitutedarabinocytidine,2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine,2′-deoxy-4-thiouridine, other non-natural pyrimidine nucleosides, or1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;

Z is guanosine or 2′-deoxyguanosine, is 2′ deoxy-7-deazaguanosine,2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2′substituted-arabinoguanosine, 2′-O-substituted-arabinoguanosine,2′-deoxyinosine, or other non-natural purine nucleoside

N1, at each occurrence, is preferably a naturally occurring or asynthetic nucleoside or an immunostimulatory moiety selected from thegroup consisting of abasic nucleosides, arabinonucleosides,2′-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, andnucleosides linked by a phosphodiester or modified internucleosidelinkage to the adjacent nucleoside on the 3′ side, the modifiedinternucleotide linkage being selected from, without limitation, alinker having a length of from about 2 angstroms to about 200 angstroms,C2-C18 alkyl linker, poly(ethylene glycol) linker,2-aminobutyl-1,3-propanediol linker, glyceryl linker, 2′-5′internucleoside linkage, and phosphorothioate, phosphorodithioate, ormethylphosphonate internucleoside linkage;

Nn, at each occurrence, is a naturally occurring nucleoside or animmunostimulatory moiety, preferably selected from the group consistingof abasic nucleosides, arabinonucleosides, 2′-deoxyuridine,α-deoxyribonucleosides, 2′-β-substituted ribonucleosides, andnucleosides linked by a modified internucleoside linkage to the adjacentnucleoside on the 3′ side, the modified internucleotide linkage beingselected from the group consisting of amino linker, C2-C18 alkyl linker,poly(ethylene glycol) linker, 2-aminobutyl-1,3-propanediol linker,glyceryl linker, 2′-5′ internucleoside linkage, and methylphosphonateinternucleoside linkage;

provided that at least one N1 or Nn is an immunostimulatory moiety;

wherein n is a number from 0-30;

wherein the 3′ nucleoside is optionally linked directly or via anon-nucleotidic linker to another oligonucleotide, which may or may notbe immunostimulatory.

In a second aspect, the invention provides a method for treating cancerin a cancer patient comprising administering an immunostimulatoryoligonucleotide and/or immunomer conjugate, which comprises animmunostimulatory oligonucleotide and/or immunomer, as described above,and a cancer antigen conjugated to the immunostimulatory oligonucleotideand/or immunomer at a position other than the accessible 5′ end, incombination with a chemotherapeutic agent.

In a third aspect, the invention provides pharmaceutical formulationscomprising an immunostimulatory oligonucleotide and/or immunostimulatoryoligonucleotide and/or an immunomer or immunomer conjugate according tothe invention, a chemotherapeutic agent and a physiologically acceptablecarrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of representative immunomers of theinvention.

FIG. 2 depicts several representative immunomers of the invention.

FIG. 3 depicts a group of representative small molecule linkers suitablefor linear synthesis of immunomers of the invention.

FIG. 4 depicts a group of representative small molecule linkers suitablefor parallel synthesis of immunomers of the invention.

FIG. 5 is a synthetic scheme for the linear synthesis of immunomers ofthe invention. DMTr=4,4′-dimethoxytrityl; CE=cyanoethyl.

FIG. 6 is a synthetic scheme for the parallel synthesis of immunomers ofthe invention. DMTr=4,4′-dimethoxytrityl; CE=cyanoethyl.

FIG. 7A is a graphic representation of the induction of IL-12 byOligonucleotide 1 and Immunomers 2-3 in BALB/c mouse spleen cellcultures. These data suggest that Immunomer 2, which has accessible5′-ends, is a stronger inducer of IL-12 than monomeric Oligo 1, and thatImmunomer 3, which does not have accessible 5′-ends, has equal or weakerability to produce immune stimulation compared with oligo 1.

FIG. 7B is a graphic representation of the induction of IL-6 (top tobottom, respectively) by Oligonucleotide 1 and Immunomers 2-3 in BALB/cmouse spleen cells cultures. These data suggest that Immunomer 2, whichhas accessible 5′-ends, is a stronger inducer of IL-6 than monomericOligo 1, and that Immunomer 3, which does not have accessible 5′-ends,has equal or weaker ability to induce immune stimulation compared withOligo 1.

FIG. 7C is a graphic representation of the induction of IL-10 byOligonucleotide 1 and Immunomers 2-3 (top to bottom, respectively) inBALB/c mouse spleen cell cultures.

FIG. 8A is a graphic representation of the induction of BALB/c mousespleen cell proliferation in cell cultures by different concentrationsof Immunomers 5 and 6, which have inaccessible and accessible 5′-ends,respectively.

FIG. 8B is a graphic representation of BALB/c mouse spleen enlargementby Oligonucleotide 4 and Immunomers 5-6, which have an immunogenicchemical modification in the 5′-flanking sequence of the CpG motif.Again, the immunomer, which has accessible 5′-ends (6), has a greaterability to increase spleen enlargement compared with Immunomer 5, whichdoes not have accessible 5′-end and with monomeric Oligonucleotide 4.

FIG. 9A is a graphic representation of induction of IL-12 by differentconcentrations of Oligonucleotide 4 and Immunomers 7 and 8 in BALB/cmouse spleen cell cultures.

FIG. 9B is a graphic representation of induction of IL-6 by differentconcentrations of Oligonucleotide 4 and Immunomers 7 and 8 in BALB/cmouse spleen cell cultures.

FIG. 9C is a graphic representation of induction of IL-10 by differentconcentrations of Oligonucleotide 4 and Immunomers 7 and 8 in BALB/cmouse spleen cell cultures.

FIG. 10A is a graphic representation of the induction of cellproliferation by Immunomers 14, 15, and 16 in BALB/c mouse spleen cellcultures.

FIG. 10B is a graphic representation of the induction of cellproliferation by IL-12 by different concentrations of Immunomers 14 and16 in BALB/c mouse spleen cell cultures.

FIG. 10C is a graphic representation of the induction of cellproliferation by IL-6 by different concentrations of Immunomers 14 and16 in BALB/c mouse spleen cell cultures.

FIG. 11A is a graphic representation of the induction of cellproliferation by Oligonucleotides 4 and 17 and Immunomers 19 and 20 inBALB/c mouse spleen cell cultures.

FIG. 11B is a graphic representation of the induction of cellproliferation IL-12 by different concentrations of Oligonucleotides 4and 17 and Immunomers 19 and 20 in BALB/c mouse spleen cell cultures.

FIG. 11C is a graphic representation of the induction of cellproliferation IL-6 by different concentrations of Oligonucleotides 4 and17 and Immunomers 19 and 20 in BALB/c mouse spleen cell cultures.

FIG. 12 is a graphic representation of BALB/c mouse spleen enlargementusing Oligonucleotide 4 and Immunomers 14, 23, and 24.

FIG. 13 shows the effect of a method according to the invention on tumorgrowth in a nude mouse model for prostate cancer.

FIG. 14 shows the effect of a method according to the invention on bodyweight of the mice used in the study.

FIG. 15 shows examples of IMO compound structures and modifications.

FIG. 16 shows in vitro cytokine induction profiles of IMO compounds.

FIG. 17. (A) Antitumor activity of IMO compounds against CT26.CL25 colontumor in BALB/c mice. IMO 1 (circles), IMO 2 (triangles) or controlnon-CpG DNA (squares). * p<0.001 compared with non-CpG DNA controlgroup. Plots showing the survival of (B) CT26.WT or (C) CT26.CL25 tumorbearing BALB/c mice in different treatment groups. PBS (squares) orcontrol non-CpG DNA (circles) or IMO 2 (triangles)

FIG. 18. Levels of serum β-gal-specific IgG1 (open bars) and IgG2a(filled bars) in CT26.CL25 tumor bearing BALB/c mice on day 24.

FIG. 19. (A) IFN-γ and (B) IL-4 secreting T-lymphocytes in total T cells(10⁶) isolated from the spleens of CT26.CL25 colon tumor bearing mice onday 24 in various treatment groups. PBS (open bars), β-gal (shadedbars), or OVA peptide (black bars).

FIG. 20. Persistent antitumor memory following IMO treatment. Survivalplots of long term survivors of IMO treated CT26.CL25 peritoneal tumorbearing mice rechallenged with (A) CT26.WT colon, (B) CT26.CL25 colon,or (C) 4T1 mammary carcinoma cells. IMO 2 (circles) or naive mice thatwere not treated with IMO motifs (squares).

FIG. 21. Naïve mice develop specific antitumor protection followingadoptive transfer of immune cells from tumor bearing mice that weretreated with IMO compounds. (A) Survival of the mice to parentalCT26.CL25 tumor cell challenge following adoptive transfer of the immunecells obtained from mice treated with IMO 2 (triangle) or naïve mice(circles). (B) Survival of the mice to 4T1 breast cancer cell challengefollowing adoptive transfer of the immune cells obtained from micetreated with IMO 2 (triangle) or naïve mice (circles). Mice injectedwith PBS and challenged with CT26.CL25 cells as control are shown insquares in both the panels.

FIG. 22. (A) Antitumor activity of IMO compounds against B16.F0 melanomain C57BL/6 mice. IMO 2 (filled bars) or control non-CpG DNA (open bars)*p<0.0183 compared with non-CpG DNA control group. Total serum (B) IgG1and (C) IgG2a antibody subclasses in B16.F0 tumor bearing C57BL/6 miceon day 22 following treatment with IMO 2 or control non-CpG DNA.

FIG. 23. Effect of IMO 2 on survival of B16.F0 melanoma bearing wt, IL-6ko, and IL-12 ko C57BL/6 mice. Non-CpG DNA to wild-type (wt) (squares)and IMO 2 to wt (diamonds), IL-6 knockout (ko) (circles) and IL-12 ko(triangles).

FIG. 24. Synergistic antitumor activity of the combination ofconventional chemotherapy and IMO immunotherapy. (A) Growth inhibitionof 4T1 breast tumor in BALB/c mice in various treatment groups. Eachcircle represents data of a single animal and + indicates average. *p=0.0004 compared with PBS control group. (B) Survival plots ofperitoneal disseminated B16.F0 melanoma bearing C57BL/6 mice in varioustreatment groups. PBS (squares), docetaxel (20 mg/kg, i.p) single doseon day 2 (diamonds), IMO 2 (2.5 mg/kg, i.p, on days 3, 6, 9, 12 and 15)(circles) or combination of docetaxel and IMO 2 at the same dose andschedule as in monotherapy (triangles). (C) Activation of CD69+ andCD86+ cells in C57BL/6 mice treated with PBS, docetaxel (Doce; 30 mg/kg,i.p, on days 1 and 3), IMO 2 (5 mg/kg, i.p, on days 1, 3, 5, and 7) ordocetaxel (Doce) and IMO 2. CD69+ (open bars) and CD86+ (filled bars)

FIG. 25. (A) Antitumor effects of mouse and human IMOs 2 and 3,respectively, against CT26.CL25 colon tumor in BALB/c mice. IMO 2(circles), IMO 3 (triangles) or non-CpG DNA (squares) (B) Serum IL-12levels in mice 4 hr after administration of IMO motifs.

FIG. 26 shows IMO 3 activation of human PBMCs induce lysis of Her-2positive BT-474 cells in the presence of Herceptin.

FIG. 27 shows the therapeutic schedule used in the Rituxan or Herceptincombination treatments with IMO compounds.

FIG. 28 shows the percentage of tumors and the number of days requiredfor tumors to reach 1.5 grams with Rituxan and/or IMO treatment.

FIG. 29 shows the percent inhibition of tumor growth after Herceptinand/or IMO treatment.

FIG. 30 shows the percent inhibition of tumor growth after Rituxanand/or IMO treatment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to the therapeutic use of immunostimulatoryoligonucleotides and/or immunomers as anti-cancer agents in combinationwith chemotherapeutic agents. The issued patents, patent applications,and references that are cited herein are hereby incorporated byreference to the same extent as if each was specifically andindividually indicated to be incorporated by reference. In the event ofinconsistencies between any teaching of any reference cited herein andthe present specification, the latter shall prevail for purposes of theinvention.

The invention provides methods for enhancing the anti-cancer effectcaused by immunostimulatory compounds used for immunotherapyapplications for the treatment of cancer. The immunomers and/orimmunostimulatory oligonucleotides of the invention can be used totreat, prevent or ameliorate the onset and/or progression of a tumor orcancer (e.g., tumors of the brain, lung (e.g., small cell and non-smallcell), ovary, breast, prostate, colon, glioma, as well as othermelanoma, carcinomas, leukemias, lymphomas and sarcomas). In the methodsaccording to the invention, immunostimulatory oligonucleotides and/orimmunomers provide a synergistic therapeutic effect when use incombination with chemotherapeutic agents. This result is surprising inview of the fact that immunostimulatory oligonucleotides and immunomerscause cell division of immune system cells, whereas chemotherapeuticagents normally kill actively dividing cells.

In a first aspect, the invention provides a method for treating cancerin a cancer patient comprising administering, in combination withchemotherapeutic agents, immunostimulatory oligonucleotides and/orimmunomers, the latter comprising at least two oligonucleotides linkedtogether, such that the immunomer has more than one accessible 5′ end,wherein at least one of the oligonucleotides is an immunostimulatoryoligonucleotide. As used herein, the term “accessible 5′ end” means thatthe 5′ end of the oligonucleotide is sufficiently available such thatthe factors that recognize and bind to immunomers and stimulate theimmune system have access to it. Optionally, the 5′OH can be linked to aphosphate, phosphorothioate, or phosphorodithioate moiety, an aromaticor aliphatic linker, cholesterol, or another entity which does notinterfere with accessibility. Immunostimulatory oligonucleotides andimmunomers induce an immune response when administered to a vertebrate.When used in combination with chemotherapeutic agents, a synergistictherapeutic effect is obtained.

Preferred chemotherapeutic agents used in the method according to theinvention include, without limitation Gemcitabine, methotrexate,vincristine, adriamycin, cisplatin, non-sugar containingchloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin,doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin,carmustaine and poliferposan, MMI270, BAY 12-9566, RAS farnesyltransferase inhibitor, farnesyl transferase inhibitor, MMP,MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470,Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone,Metaret/Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340,AG3433, Incel/VX-710, VX-853, ZD0101, IS1641, ODN 698, TA2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f,Lemonal DP 2202, FK 317, imatinib mesylate/Gleevec, Picibanil/OK-432, AD32/Valrubicin, Metastron/strontium derivative, Temodal/Temozolomide,Evacet/liposomal doxorubicin, Yewtaxan/Placlitaxel, Taxol/Paclitaxel,Xeload/Capecitabine, Furtulon/Doxifluridine, Cyclopax/oral paclitaxel,Oral Taxoid, SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358(774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, BMS-182751/oralplatinum, UFT (Tegafur/Uracil), Ergamisol/Levamisole,Eniluracil/776C85/5FU enhancer, Campto/Levamisole, Camptosar/Irinotecan,Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel,Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin,Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU79553/Bis-Naphtalimide, LU 103793/Dolastain, Caetyx/liposomaldoxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine seeds,CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide,Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin,Plantinol/cisplatin, Vepeside/Etoposide, ZD 9331, Taxotere/Docetaxel,prodrug of guanine arabinoside, Taxane Analog, nitrosoureas, alkylatingagents such as melphelan and cyclophosphamide, Aminoglutethimide,Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl,Dactinomycin, Daunorubicin HCl, Estramustine phosphate sodium, Etoposide(VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolideacetate (LHRH-releasing factor analogue), Lomustine (CCNU),Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna, Mitotane(o.p′-DDD), Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl,Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastinesulfate, Amsacrine (m-AMSA), Azacitidine, Erthropoietin,Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG; methylglyoxal bis-guanylhydrazone; MGBG), Pentostatin (2′ deoxycoformycin),Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine sulfate.

In the methods according to this aspect of the invention, administrationof immunostimulatory oligonucleotides and/or immunomers can be by anysuitable route, including, without limitation, parenteral, oral,sublingual, transdermal, topical, intranasal, aerosol, intraocular,intratracheal, intrarectal, vaginal, by gene gun, dermal patch or in eyedrop or mouthwash form. Administration of the therapeutic compositionsof immunostimulatory oligonucleotides and/or immunomers can be carriedout using known procedures at dosages and for periods of time effectiveto reduce symptoms or surrogate markers of the disease. Whenadministered systemically, the therapeutic composition is preferablyadministered at a sufficient dosage to attain a blood level ofimmunostimulatory oligonucleotide and/or immunomer from about 0.0001micromolar to about 10 micromolar. For localized administration, muchlower concentrations than this may be effective, and much higherconcentrations may be tolerated. Preferably, a total dosage ofimmunostimulatory oligonucleotide and/or immunomer ranges from about0.0001 mg per patient per day to about 200 mg per kg body weight perday. It may be desirable to administer simultaneously, or sequentially atherapeutically effective amount of one or more of the therapeuticcompositions of the invention to an individual as a single treatmentepisode.

For purposes of this aspect of the invention, the term “in combinationwith” means in the course of treating the same disease in the samepatient, and includes administering the immunostimulatoryoligonucleotide and/or immunomer and/or the chemotherapeutic agent inany order, including simultaneous administration, as well as temporallyspaced order of up to several days apart. Such combination treatment mayalso include more than a single administration of the immunostimulatoryoligonucleotide and/or immunomer, and/or independently thechemotherapeutic agent. The administration of the immunostimulatoryoligonucleotide and/or immunomer and/or chemotherapeutic agent may be bythe same or different routes.

In some embodiments, the immunomer used in the method according to theinvention comprises two or more immunostimulatory oligonucleotides, (inthe context of the immunomer) which may be the same or different.Preferably, each such immunostimulatory oligonucleotide has at least oneaccessible 5′ end.

In certain embodiments of the method according to the invention, inaddition to the immunostimulatory oligonucleotide(s), the immunomer alsocomprises at least one oligonucleotide that is complementary to a gene.As used herein, the term “complementary to” means that theoligonucleotide hybridizes under physiological conditions to a region ofthe gene. In some embodiments, the oligonucleotide downregulatesexpression of a gene. Such downregulatory oligonucleotides preferablyare selected from the group consisting of antisense oligonucleotides,ribozyme oligonucleotides, small inhibitory RNAs and decoyoligonucleotides. As used herein, the term “downregulate a gene” meansto inhibit the transcription of a gene or translation of a gene product.Thus, the immunomers used in the method according to the invention canbe used to target one or more specific disease targets, while alsostimulating the immune system.

In certain embodiments, the immunostimulatory oligonucleotide and/orimmunomer used in the method according to the invention includes aribozyme or a decoy oligonucleotide. As used herein, the term “ribozyme”refers to an oligonucleotide that possesses catalytic activity.Preferably, the ribozyme binds to a specific nucleic acid target andcleaves the target. As used herein, the term “decoy oligonucleotide”refers to an oligonucleotide that binds to a transcription factor in asequence-specific manner and arrests transcription activity. Preferably,the ribozyme or decoy oligonucleotide exhibits secondary structure,including, without limitation, stem-loop or hairpin structures. Incertain embodiments, at least one oligonucleotide comprisespoly(I)-poly(dC). In certain embodiments, at least one set of Nnincludes a string of 3 to 10 dGs and/or Gs or 2′-substituted ribo orarabino Gs.

For purposes of the invention, the term “oligonucleotide” refers to apolynucleoside formed from a plurality of linked nucleoside units. Sucholigonucleotides can be obtained from existing nucleic acid sources,including genomic or cDNA, but are preferably produced by syntheticmethods. In preferred embodiments each nucleoside unit includes aheterocyclic base and a pentofuranosyl, trehalose, arabinose,2′-deoxy-2′-substituted arabinose, 2′-O-substituted arabinose or hexosesugar group. The nucleoside residues can be coupled to each other by anyof the numerous known internucleoside linkages. Such internucleosidelinkages include, without limitation, phosphodiester, phosphorothioate,phosphorodithioate, alkylphosphonate, alkylphosphonothioate,phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy,acetamidate, carbamate, morpholino, borano, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridgedphosphorothioate, and sulfone internucleoside linkages. The term“oligonucleotide” also encompasses polynucleosides having one or morestereospecific internucleoside linkage (e.g., (R_(P))- or(S_(P))-phosphorothioate, alkylphosphonate, or phosphotriesterlinkages). As used herein, the terms “oligonucleotide” and“dinucleotide” are expressly intended to include polynucleosides anddinucleosides having any such internucleoside linkage, whether or notthe linkage comprises a phosphate group. In certain preferredembodiments, these internucleoside linkages may be phosphodiester,phosphorothioate, phosphorodithioate, methylphosphonate linkages, orcombinations thereof.

In some embodiments, the immunomer comprises oligonucleotides eachhaving from about 3 to about 35 nucleoside residues, preferably fromabout 4 to about 30 nucleoside residues, more preferably from about 4 toabout 20 nucleoside residues. In some embodiments, the oligonucleotideshave from about 5 or 6 to about 18, or from about 5 or 6 to about 14,nucleoside residues. As used herein, the term “about” implies that theexact number is not critical. Thus, the number of nucleoside residues inthe oligonucleotides is not critical, and oligonucleotides having one ortwo fewer nucleoside residues, or from one to several additionalnucleoside residues are contemplated as equivalents of each of theembodiments described above, for purposes of this invention. In someembodiments, one or more of the oligonucleotides have 11 nucleotides.

The term “oligonucleotide” also encompasses polynucleosides havingadditional substituents including, without limitation, protein groups,lipophilic groups, intercalating agents, diamines, folic acid,cholesterol and adamantane. The term “oligonucleotide” also encompassesany other nucleobase containing polymer, including, without limitation,peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups(PHONA), locked nucleic acids (LNA), morpholino-backboneoligonucleotides, and oligonucleotides having backbone sections withalkyl linkers or amino linkers.

The immunostimulatory oligonucleotides and/or immunomers used in themethod according to the invention can include naturally occurringnucleosides, modified nucleosides, or mixtures thereof. As used herein,the term “modified nucleoside” is a nucleoside that includes a modifiedheterocyclic base, a modified sugar moiety, or a combination thereof. Insome embodiments, the modified nucleoside is a non-natural pyrimidine orpurine nucleoside, as herein described. In some embodiments, themodified nucleoside is a 2′-substituted ribonucleoside anarabinonucleoside or a 2′-deoxy-2′-fluoroarabinoside.

For purposes of the invention, the term “2′-substituted ribonucleoside”includes ribonucleosides in which the hydroxyl group at the 2′ positionof the pentose moiety is substituted to produce a 2′-O-substitutedribonucleoside. Preferably, such substitution is with a lower alkylgroup containing 1-6 saturated or unsaturated carbon atoms, or with anaryl group having 6-10 carbon atoms, wherein such alkyl, or aryl groupmay be unsubstituted or may be substituted, e.g., with halo, hydroxy,trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carboalkoxy, or amino groups. Examples of such 2′-O-substitutedribonucleosides include, without limitation 2′-O-methylribonucleosidesand 2′-O-methoxyethylribonucleosides.

The term “2′-substituted ribonucleoside” also includes ribonucleosidesin which the 2′-hydroxyl group is replaced with a lower alkyl groupcontaining 1-6 saturated or unsaturated carbon atoms, or with an aminoor halo group. Examples of such 2′-substituted ribonucleosides include,without limitation, 2′-amino, 2′-fluoro, 2′-allyl, and 2′-propargylribonucleosides.

The term “oligonucleotide” includes hybrid and chimericoligonucleotides. A “chimeric oligonucleotide” is an oligonucleotidehaving more than one type of internucleoside linkage. One preferredexample of such a chimeric oligonucleotide is a chimeric oligonucleotidecomprising a phosphorothioate, phosphodiester or phosphorodithioateregion and non-ionic linkages such as alkylphosphonate oralkylphosphonothioate linkages (see e.g., Pederson et al. U.S. Pat. Nos.5,635,377 and 5,366,878).

A “hybrid oligonucleotide” is an oligonucleotide having more than onetype of nucleoside. One preferred example of such a hybridoligonucleotide comprises a ribonucleotide or 2′-substitutedribonucleotide region, and a deoxyribonucleotide region (see, e.g.,Metelev and Agrawal, U.S. Pat. Nos. 5,652,355, 6,346,614 and 6,143,881).

For purposes of the invention, the term “immunostimulatoryoligonucleotide” refers to an oligonucleotide as described above thatinduces an immune response when administered to a vertebrate, such as afish, bird, or mammal. As used herein, the term “mammal” includes,without limitation rats, mice, cats, dogs, horses, cattle, cows, pigs,rabbits, non-human primates, and humans. Useful immunostimulatoryoligonucleotides can be found described in Agrawal et al., WO 98/49288,published Nov. 5, 1998; WO 01/12804, published Feb. 22, 2001; WO01/55370, published Aug. 2, 2001; PCT/US01/13682, filed Apr. 30, 2001;and PCT/US01/30137, filed Sep. 26, 2001. Preferably, theimmunostimulatory oligonucleotide comprises at least one phosphodiester,phosphorothioate, methylphosphonate, or phosphordithioateinternucleoside linkage.

In some embodiments, at least one immunostimulatory oligonucleotide ofthe immunomer comprises an immunostimulatory dinucleotide of formula5′-Pyr-Pur-3′, wherein Pyr is a natural or synthetic pyrimidinenucleoside and Pur is a natural or synthetic purine nucleoside. As usedherein, the term “pyrimidine nucleoside” refers to a nucleoside whereinthe base component of the nucleoside is a pyrimidine base. Similarly,the term “purine nucleoside” refers to a nucleoside wherein the basecomponent of the nucleoside is a purine base. For purposes of theinvention, a “synthetic” pyrimidine or purine nucleoside includes anon-naturally occurring pyrimidine or purine base, a non-naturallyoccurring sugar moiety, or a combination thereof.

Preferred pyrimidine nucleosides in the immunostimulatoryoligonucleotides and/or immunomers used in the method according to theinvention have the structure (I):

(i) wherein:

D is a hydrogen bond donor;

D′ is selected from the group consisting of hydrogen, hydrogen bonddonor, hydrogen bond acceptor, hydrophilic group, hydrophobic group,electron withdrawing group and electron donating group;

A is a hydrogen bond acceptor or a hydrophilic group;

A′ is selected from the group consisting of hydrogen bond acceptor,hydrophilic group, hydrophobic group, electron withdrawing group andelectron donating group;

X is carbon or nitrogen; and

S′ is a pentose or hexose sugar ring, or a non-naturally occurringsugar.

Preferably, the sugar ring is derivatized with a phosphate moiety,modified phosphate moiety, or other linker moiety suitable for linkingthe pyrimidine nucleoside to another nucleoside or nucleoside analog.

Preferred hydrogen bond donors include, without limitation, —NH—, —NH₂,—SH and —OH. Preferred hydrogen bond acceptors include, withoutlimitation, C═O, C═S, and the ring nitrogen atoms of an aromaticheterocycle, e.g., N3 of cytosine.

In some embodiments, the base moiety in (I) is a non-naturally occurringpyrimidine base. Examples of preferred non-naturally occurringpyrimidine bases include, without limitation, 5-hydroxycytosine,5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine,and 4-thiouracil. In some embodiments, the sugar moiety S′ in (I) is anon-naturally occurring sugar moiety. For purposes of the presentinvention, a “naturally occurring sugar moiety” is a sugar moiety thatoccurs naturally as part of nucleic acid, e.g., ribose and2′-deoxyribose, and a “non-naturally occurring sugar moiety” is anysugar that does not occur naturally as part of a nucleic acid, but whichcan be used in the backbone for an oligonucleotide, e.g, hexose.Arabinose and arabinose derivatives are examples of preferred sugarmoieties.

Preferred purine nucleoside analogs in immunostimulatoryoligonucleotides and/or immunomers used in the method according to theinvention have the structure (II):

(ii) wherein:

D is a hydrogen bond donor;

D′ is selected from the group consisting of hydrogen, hydrogen bonddonor, and hydrophilic group;

A is a hydrogen bond acceptor or a hydrophilic group;

X is carbon or nitrogen;

each L is independently selected from the group consisting of C, O, Nand S; and

S′ is a pentose or hexose sugar ring, or a non-naturally occurringsugar.

Preferably, the sugar ring is derivatized with a phosphate moiety,modified phosphate moiety, or other linker moiety suitable for linkingthe pyrimidine nucleoside to another nucleoside or nucleoside analog.

Preferred hydrogen bond donors include, without limitation, —NH—, —NH₂,—SH and —OH. Preferred hydrogen bond acceptors include, withoutlimitation, C═O, C═S, —NO₂ and the ring nitrogen atoms of an aromaticheterocycle, e.g., N1 of guanine.

In some embodiments, the base moiety in (II) is a non-naturallyoccurring purine base. Examples of preferred non-naturally occurringpurine bases include, without limitation, 6-thioguanine and7-deazaguanine. In some embodiments, the sugar moiety S′ in (II) is anaturally occurring sugar moiety, as described above for structure (I).

In preferred embodiments, the immunostimulatory dinucleotide in theimmunostimulatory oligonucleotides and/or immunomer used in the methodaccording to the invention is selected from the group consisting of CpG,C*pG, CpG*, and C*pG*, wherein C is cytidine or 2′-deoxycytidine, C* is2′-deoxythymidine, arabinocytidine, 2′-deoxythymidine,2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substitutedarabinocytidine,2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine,2′-deoxy-4-thiouridine, other non-natural pyrimidine nucleosides, or1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; G isguanosine or 2′-deoxyguanosine, G* is 2′ deoxy-7-deazaguanosine,2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2′substituted-arabinoguanosine, 2′-O-substituted-arabinoguanosine,2′-deoxyinosine, or other non-natural purine nucleoside, and p is aninternucleoside linkage selected from the group consisting ofphosphodiester, phosphorothioate, and phosphorodithioate. In certainpreferred embodiments, the immunostimulatory dinucleotide is not CpG.

The immunostimulatory oligonucleotides may include immunostimulatorymoieties on one or both sides of the immunostimulatory dinucleotide.Thus, in some embodiments, the immunostimulatory oligonucleotidecomprises an immunostimulatory domain of structure (III):

5′-Nn-N1-Y—Z—N1-Nn-3′  (III)

wherein:

Y is cytidine, 2′ deoxythymidine, 2′ deoxycytidine arabinocytidine,2′-deoxy-2′-substitutedarabinocytidine, 2′-deoxythymidine,2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine,2′-deoxy-N4-alkyl-cytidine, 2′-deoxy-4-thiouridine, other non-naturalpyrimidine nucleosides, or1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;

Z is guanosine or 2′-deoxyguanosine, 2′ deoxy-7-deazaguanosine,2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2′substituted-arabinoguanosine, 2′-O-substituted-arabinoguanosine, 2′deoxyinosine, or other non-natural purine nucleoside;

N1, at each occurrence, is preferably a naturally occurring or asynthetic nucleoside or an immunostimulatory moiety selected from thegroup consisting of abasic nucleosides, arabinonucleosides,2′-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, andnucleosides linked by a phosphodiester or modified internucleosidelinkage to the adjacent nucleoside on the 3′ side, the modifiedinternucleotide linkage being selected from, without limitation, alinker having a length of from about 2 angstroms to about 200 angstroms,C2-C18 alkyl linker, poly(ethylene glycol) linker,2-aminobutyl-1,3-propanediol linker, glyceryl linker, 2′-5′internucleoside linkage, and phosphorothioate, phosphorodithioate, ormethylphosphonate internucleoside linkage;

Nn, at each occurrence, is preferably a naturally occurring nucleosideor an immunostimulatory moiety selected from the group consisting ofabasic nucleosides, arabinonucleosides, 2′-deoxyuridine,α-deoxyribonucleosides, 2′-O-substituted ribonucleosides, andnucleosides linked by a modified internucleoside linkage to the adjacentnucleoside on the 3′ side, the modified internucleoside linkagepreferably being selected from the group consisting of amino linker,C2-C18 alkyl linker, poly(ethylene glycol) linker,2-aminobutyl-1,3-propanediol linker, glyceryl linker, 2′-5′internucleoside linkage, and methylphosphonate internucleoside linkage;

provided that at least one N1 or Nn is an immunostimulatory moiety;

wherein each n is independently a number from 0 to 30; and

wherein, in the case of an immunomer, the 3′ end is linked directly orvia a non-nucleotidic linker to another oligonucleotide, which may ormay not be immunostimulatory.

In some preferred embodiments, YZ is arabinocytidine or2′-deoxy-2′-substituted arabinocytidine and arabinoguanosine or 2′deoxy-2′-substituted arabinoguanosine. Preferred immunostimulatorymoieties include modifications in the phosphate backbones, including,without limitation, methylphosphonates, methylphosphonothioates,phosphotriesters, phosphothiotriesters, phosphorothioates,phosphorodithioates, triester prodrugs, sulfones, sulfonamides,sulfamates, formacetal, N-methylhydroxylamine, carbonate, carbamate,morpholino, boranophosphonate, phosphoramidates, especially primaryamino-phosphoramidates, N3 phosphoramidates and N5 phosphoramidates, andstereospecific linkages (e.g., (R_(P))- or (S_(P))-phosphorothioate,alkylphosphonate, or phosphotriester linkages).

Preferred immunostimulatory moieties according to the invention furtherinclude nucleosides having sugar modifications, including, withoutlimitation, 2′-substituted pentose sugars including, without limitation,2′-O-methylribose, 2′-O-methoxyethylribose, 2′-O-propargylribose, and2′-deoxy-2′-fluororibose; 3′-substituted pentose sugars, including,without limitation, 3′-O-methylribose; 1′,2′-dideoxyribose; arabinose;substituted arabinose sugars, including, without limitation,1′-methylarabinose, 3′-hydroxymethylarabinose,4′-hydroxymethyl-arabinose, and 2′-substituted arabinose sugars; hexosesugars, including, without limitation, 1,5-anhydrohexitol; andalpha-anomers. In embodiments in which the modified sugar is a3′-deoxyribonucleoside or a 3′-O-substituted ribonucleoside, theimmunostimulatory moiety is attached to the adjacent nucleoside by wayof a 2′-5′ internucleoside linkage.

Preferred immunostimulatory moieties in immunostimulatoryoligonucleotides and/or immunomers used in the method according to theinvention further include oligonucleotides having other carbohydratebackbone modifications and replacements, including peptide nucleic acids(PNA), peptide nucleic acids with phosphate groups (PHONA), lockednucleic acids (LNA), morpholino backbone oligonucleotides, andoligonucleotides having backbone linker sections having a length of fromabout 2 angstroms to about 200 angstroms, including without limitation,alkyl linkers or amino linkers. The alkyl linker may be branched orunbranched, substituted or unsubstituted, and chirally pure or a racemicmixture. Most preferably, such alkyl linkers have from about 2 to about18 carbon atoms. In some preferred embodiments such alkyl linkers havefrom about 3 to about 9 carbon atoms. Some alkyl linkers include one ormore functional groups selected from the group consisting of hydroxy,amino, thiol, thioether, ether, amide, thioamide, ester, urea, andthioether. Some such functionalized alkyl linkers are poly(ethyleneglycol) linkers of formula —O—(CH₂—CH₂—O—), (n=1-9). Some otherfunctionalized alkyl linkers are peptides or amino acids.

Preferred immunostimulatory moieties in immunostimulatoryoligonucleotides and/or immunomers used in the method according to theinvention further include DNA isoforms, including, without limitation,13-L-deoxyribonucleosides and α-deoxyribonucleosides. Preferredimmunostimulatory moieties incorporate 3′ modifications, and furtherinclude nucleosides having unnatural internucleoside linkage positions,including, without limitation, 2′-5′,2′-2′,3′-3′ and 5′-5′ linkages.

Preferred immunostimulatory moieties in immunostimulatoryoligonucleotides and/or immunomers used in the method according to theinvention further include nucleosides having modified heterocyclicbases, including, without limitation, 5-hydroxycytosine,5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine,4-thiouracil, 6-thioguanine, 7-deazaguanine, inosine, nitropyrrole,C5-propynylpyrimidine, and diaminopurines, including, withoutlimitation, 2,6-diaminopurine.

By way of specific illustration and not by way of limitation, forexample, in the immunostimulatory domain of structure (III), amethylphosphonate internucleoside linkage at position N1 or Nn is animmunostimulatory moiety, a linker having a length of from about 2angstroms to about 200 angstroms, C2-C18 alkyl linker at position X1 isan immunostimulatory moiety, and a β-L-deoxyribonucleoside at positionX1 is an immunostimulatory moiety. See Table 1 below for representativepositions and structures of immunostimulatory moieties. It is to beunderstood that reference to a linker as the immunostimulatory moiety ata specified position means that the nucleoside residue at that positionis substituted at its 3′-hydroxyl with the indicated linker, therebycreating a modified internucleoside linkage between that nucleosideresidue and the adjacent nucleoside on the 3′ side. Similarly, referenceto a modified internucleoside linkage as the immunostimulatory moiety ata specified position means that the nucleoside residue at that positionis linked to the adjacent nucleoside on the 3′ side by way of therecited linkage.

TABLE 1 Position TYPICAL IMMUNOSTIMULATORY MOIETIES N1Naturally-occurring nucleosides, abasic nucleoside, arabinonucleoside,2′-deoxyuridine, β-L- deoxyribonucleoside C2-C18 alkyl linker,poly(ethylene glycol) linkage, 2-aminobutyl-1,3-propanediol linker(amino linker), 2′-5′ internucleoside linkage, methylphosphonateinternucleoside linkage Nn Naturally-occurring nucleosides, abasicnucleoside, arabinonucleosides, 2′-deoxyuridine, 2′-O-substitutedribonucleoside, 2′-5′ internucleoside linkage, methylphosphonateinternucleoside linkage, provided that N1 and N2 cannot both be abasiclinkages

Table 2 shows representative positions and structures ofimmunostimulatory moieties within an immunostimulatory oligonucleotidehaving an upstream potentiation domain. As used herein, the term “Spacer9” refers to a poly(ethylene glycol) linker of formula—O—(CH₂CH₂—O)_(n)—, wherein n is 3. The term “Spacer 18” refers to apoly(ethylene glycol) linker of formula —O—(CH₂CH₂—O)_(n)—, wherein n is6. As used herein, the term “C2-C18 alkyl linker refers to a linker offormula —O—(CH₂)_(q)—O—, where q is an integer from 2 to 18.Accordingly, the terms “C3-linker” and “C3-alkyl linker” refer to alinker of formula —O—(CH₂)₃—O—. For each of Spacer 9, Spacer 18, andC2-C18 alkyl linker, the linker is connected to the adjacent nucleosidesby way of phosphodiester, phosphorothioate, phosphorodithioate ormethylphosphonate linkages.

TABLE 2 Position TYPICAL IMMUNOSTIMULATORY MOIETY 5′ N2Naturally-occurring nucleosides, 2-aminobutyl-1,3-propanediol linker 5′N1 Naturally-occurring nucleosides, β-L-deoxyribonucleoside, C2-C18alkyl linker, poly(ethylene glycol), abasic linker,2-aminobutyl-1,3-propanediol linker 3′ N1 Naturally-occurringnucleosides, 1′,2′-dideoxyribose, 2′-O-methyl- ribonucleoside, C2-C18alkyl linker, Spacer 9, Spacer 18 3′ N2 Naturally-occurring nucleosides,1′,2′-dideoxyribose, 3′- deoxyribonucleoside, β-L-deoxyribonucleoside,2′-O-propargyl- ribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer18, methylphosphonate internucleoside linkage 3′ N 3 Naturally-occurringnucleosides, 1′,2′-dideoxyribose, C2-C18 alkyl linker, Spacer 9, Spacer18, methylphosphonate internucleoside linkage, 2′-5′ internucleosidelinkage, d(G)n, polyI-polydC 3′N 2 + 3′N 3 1′,2′-dideoxyribose,β-L-deoxyribonucleoside, C2-C18 alkyl linker, d(G)n, polyI-polydC 3′N3 +3′ N 4 2′-O-methoxyethyl-ribonucleoside, methylphosphonateinternucleoside linkage, d(G)n, polyI-polydC 3′N5 + 3′ N 61′,2′-dideoxyribose, C2-C18 alkyl linker, d(G)n, polyI-polydC 5′N1 + 3′N 3 1′,2′-dideoxyribose, d(G)n, polyI-polydC

Table 3 shows representative positions and structures ofimmunostimulatory moieties within an immunostimulatory oligonucleotidehaving a downstream potentiation domain.

TABLE 3 Position TYPICAL IMMUNOSTIMULATORY MOIETY 5′ N2methylphosphonate internucleoside linkage 5′ N1 methylphosphonateinternucleoside linkage 3′ N1 1′,2′-dideoxyribose, methylphosphonateinternucleoside linkage, 2′-O-methyl 3′ N2 1′,2′-dideoxyribose,β-L-deoxyribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer 18,2-aminobutyl-1,3-propanediol linker, methylphosphonate internucleosidelinkage, 2′-O-methyl 3′ N3 3′-deoxyribonucleoside, 3′-O-substitutedribonucleoside, 2′-O-propargyl-ribonucleoside 3′N2 + 3′ N31′,2′-dideoxyribose, β-L-deoxyribonucleoside

The immunomers used in the method according to the invention comprise atleast two oligonucleotides linked directly or via a non-nucleotidiclinker. For purposes of the invention, a “non-nucleotidic linker” is anymoiety that can be linked to the oligonucleotides by way of covalent ornon-covalent linkages. Preferably such linker is from about 2 angstromsto about 200 angstroms in length. Several examples of preferred linkersare set forth below. Non-covalent linkages include, but are not limitedto, electrostatic interaction, hydrophobic interactions, π-stackinginteractions, and hydrogen bonding. The term “non-nucleotidic linker” isnot meant to refer to an internucleoside linkage, as described above,e.g., a phosphodiester, phosphorothioate, or phosphorodithioatefunctional group, that directly connects the 3′-hydroxyl groups of twonucleosides. For purposes of this invention, such a direct 3′-3′ linkageis considered to be a “nucleotidic linkage.”

In some embodiments, the non-nucleotidic linker is a metal, including,without limitation, gold particles. In some other embodiments, thenon-nucleotidic linker is a soluble or insoluble biodegradable polymerbead.

In yet other embodiments, the non-nucleotidic linker is an organicmoiety having functional groups that permit attachment to theoligonucleotide. Such attachment preferably is by any stable covalentlinkage.

In some embodiments, the non-nucleotidic linker is a biomolecule,including, without limitation, polypeptides, antibodies, lipids,antigens, allergens, and oligosaccharides. In some other embodiments,the non-nucleotidic linker is a small molecule. For purposes of theinvention, a small molecule is an organic moiety having a molecularweight of less than 1,000 Da. In some embodiments, the small moleculehas a molecular weight of less than 750 Da.

In some embodiments, the small molecule is an aliphatic or aromatichydrocarbon, either of which optionally can include, either in thelinear chain connecting the oligonucleotides or appended to it, one ormore functional groups selected from the group consisting of hydroxy,amino, thiol, thioether, ether, amide, thioamide, ester, urea, andthiourea. The small molecule can be cyclic or acyclic. Examples of smallmolecule linkers include, but are not limited to, amino acids,carbohydrates, cyclodextrins, adamantane, cholesterol, haptens andantibiotics. However, for purposes of describing the non-nucleotidiclinker, the term “small molecule” is not intended to include anucleoside.

In some embodiments, the small molecule linker is glycerol or a glycerolhomolog of the formula HO—(CH₂)_(o)—CH(OH)—(CH₂)_(p)—OH, wherein o and pindependently are integers from 1 to about 6, from 1 to about 4, or from1 to about 3. In some other embodiments, the small molecule linker is aderivative of 1,3-diamino-2-hydroxypropane. Some such derivatives havethe formula HO—(CH₂)_(m)—C(O)NH—CH₂—CH(OH)—CH₂—NHC(O)—(CH₂)_(m)—OH,wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 toabout 6, or from 2 to about 4.

Some non-nucleotidic linkers in immunomers used in the method accordingto the invention permit attachment of more than two oligonucleotides, asschematically depicted in FIG. 1. For example, the small molecule linkerglycerol has three hydroxyl groups to which oligonucleotides may becovalently attached. Some immunomers according to the invention,therefore, comprise more than two oligonucleotides linked at their 3′ends to a non-nucleotidic linker. Some such immunomers comprise at leasttwo immunostimulatory oligonucleotides, each having an accessible 5′end.

The immunostimulatory oligonucleotides and/or immunomers used in themethod according to the invention may conveniently be synthesized usingan automated synthesizer and phosphoramidite approach as schematicallydepicted in FIGS. 5 and 6, and further described in the Examples. Insome embodiments, the immunostimulatory oligonucleotides and/orimmunomers are synthesized by a linear synthesis approach (see FIG. 5).As used herein, the term “linear synthesis” refers to a synthesis thatstarts at one end of the immunomer and progresses linearly to the otherend. Linear synthesis permits incorporation of either identical orun-identical (in terms of length, base composition and/or chemicalmodifications incorporated) monomeric units into the immunostimulatoryoligonucleotides and/or immunomers.

An alternative mode of synthesis for immunomers is “parallel synthesis”,in which synthesis proceeds outward from a central linker moiety (seeFIG. 6). A solid support attached linker can be used for parallelsynthesis, as is described in U.S. Pat. No. 5,912,332. Alternatively, auniversal solid support, such as phosphate attached to controlled poreglass support, can be used.

Parallel synthesis of immunomers has several advantages over linearsynthesis: (1) parallel synthesis permits the incorporation of identicalmonomeric units; (2) unlike in linear synthesis, both (or all) themonomeric units are synthesized at the same time, thereby the number ofsynthetic steps and the time required for the synthesis is the same asthat of a monomeric unit; and (3) the reduction in synthetic stepsimproves purity and yield of the final immunomer product.

At the end of the synthesis by either linear synthesis or parallelsynthesis protocols, the immunostimulatory oligonucleotides orimmunomers used in the method according to the invention mayconveniently be deprotected with concentrated ammonia solution or asrecommended by the phosphoramidite supplier, if a modified nucleoside isincorporated. The product immunostimulatory oligonucleotides and/orimmunomer is preferably purified by reversed phase HPLC, detritylated,desalted and dialyzed.

Table 4 shows representative immunomers used in the method according tothe invention. Additional immunomers are found described in theExamples.

TABLE 4 Examples of Immunomer Sequences Oligo or Immunomer No. Sequencesand Modification (5′-3′) 1 5′-GAGAACGCTCGACCTT-3′ 25′-GAGAACGCTCGACCTT-3′-3′-TTCCAGCTCGCAAGAG-5′ 33′-TTCCAGCTCGCAAGAG-5′-5′-GAGAACGCTCGACCTT-3′ 4 5′-CTATCTGACGTTCTCTGT-3′5

6

7

8

9

10

11

12

13 5′-CTGACGTTCTCTGT-3′ 14

15

16

17 5′-XXTGACGTTCTCTGT-3′ 18

19

20

21 5′-TCTGACGTTCT-3′ 22

23

24

L = C3-alkyl linker; X = 1′,2′-dideoxyriboside; Y = ^(5OH)dC; R =7-deaza-dG

In a second aspect, the invention provides a method for treating cancerin a cancer patient comprising administering to the patient achemotherapeutic agent in combination with an immunostimulatoryoligonucleotide and/or immunomer conjugate, which comprises animmunostimulatory oligonucleotide and/or immunomer, as described above,and an antigen conjugated to the immunostimulatory oligonucleotideand/or immunomer at a position other than the accessible 5′ end. In someembodiments, the non-nucleotidic linker comprises an antigen associatedwith cancer, which is conjugated to the oligonucleotide. In some otherembodiments, the antigen is conjugated to the oligonucleotide at aposition other than its 3′ end. In some embodiments, the antigenproduces a vaccine effect. For purposes of the invention, the term“associated with” means that the antigen is present when the cancer ispresent, but either is not present, or is present in reduced amounts,when the cancer is absent.

The immunostimulatory oligonucleotides and/or immunomer is covalentlylinked to the antigen, or it is otherwise operatively associated withthe antigen. As used herein, the term “operatively associated with”refers to any association that maintains the activity of bothimmunostimulatory oligonucleotide and/or immunomer and antigen.Nonlimiting examples of such operative associations include being partof the same liposome or other such delivery vehicle or reagent.Additionally, a nucleic acid molecule encoding the antigen can be clonedinto an expression vector and administered in combination with theimmunostimulatory oligonucleotide and/or immunomer. As used herein, theterm “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. Preferred vectors arethose capable of autonomous replication and expression of nucleic acidsto which they are linked (e.g., an episome). Vectors capable ofdirecting the expression of genes to which they are operatively linkedare referred to herein as “expression vectors.” In general, expressionvectors of utility in recombinant DNA techniques are often in the formof “plasmids” which refer generally to circular double stranded DNAloops which, in their vector form, are not bound to the chromosome. Inthe present specification, “plasmid” and “vector” are usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors which serve equivalent functions and which becomeknown in the art subsequently hereto.

In embodiments wherein the immunostimulatory oligonucleotide and/orimmunomer is covalently linked to the antigen, such covalent linkagepreferably is at any position on the immunostimulatory oligonucleotideand/or immunomer other than an accessible 5′ end of an immunostimulatoryoligonucleotide. For example, the antigen may be attached at aninternucleoside linkage or may be attached to the non-nucleotidiclinker. Alternatively, the antigen may itself be the non-nucleotidiclinker.

In a third aspect, the invention provides pharmaceutical formulationscomprising an immunostimulatory oligonucleotide and/or immunostimulatoryoligonucleotide conjugate and/or immunomer or immunomer conjugateaccording to the invention, a chemotherapeutic agent and aphysiologically acceptable carrier. As used herein, the term“physiologically acceptable” refers to a material that does notinterfere with the effectiveness of the immunomer and is compatible witha biological system such as a cell, cell culture, tissue, or organism.Preferably, the biological system is a living organism, such as avertebrate. Preferred chemotherapeutic agents include, withoutlimitation Gemcitabine methotrexate, vincristine, adriamycin, cisplatin,non-sugar containing chloroethylnitrosoureas, 5-fluorouracil, mitomycinC, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA,valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566, RASfarnesyl transferase inhibitor, farnesyl transferase inhibitor, MMP,MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470,Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone,Metaret/Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340,AG3433, Incel/VX-710, VX-853, ZD0101, IS1641, ODN 698, TA2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f,Lemonal DP 2202, FK 317, imatinib mesylate/Gleevec, imatinibmesylate/Gleevec, Picibanil/OK-432, AD 32/Valrubicin,Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomaldoxorubicin, Yewtaxan/Placlitaxel, Taxol/Paclitaxel,Xeload/Capecitabine, Furtulon/Doxifluridine, Cyclopax/oral paclitaxel,Oral Taxoid, SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358(774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, BMS-182751/oralplatinum, UFT (Tegafur/Uracil), Ergamisol/Levamisole,Eniluracil/776C85/5FU enhancer, Campto/Levamisole, Camptosar/Irinotecan,Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel,Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin,Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU79553/Bis-Naphtalimide, LU 103793/Dolastain, Caetyx/liposomaldoxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, iodine seeds,CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide,Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin,Plantinol/cisplatin, Vepeside/Etoposide, ZD 9331, Taxotere/Docetaxel,prodrug of guanine arabinoside, Taxane Analog, nitrosoureas, alkylatingagents such as melphelan and cyclophosphamide, Aminoglutethimide,Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl,Dactinomycin, Daunorubicin HCl, Estramustine phosphate sodium, Etoposide(VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolideacetate (LHRH-releasing factor analogue), Lomustine (CCNU),Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna, Mitotane(o.p′-DDD), Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl,Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastinesulfate, Amsacrine (m-AMSA), Azacitidine, Erthropoietin,Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG; methylglyoxal bis-guanylhydrazone; MGBG), Pentostatin (2′ deoxycoformycin),Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine sulfate.

In yet another embodiment, the formulations include a cancer vaccineselected from the group consisting of EFG, Anti-idiotypic cancervaccines, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugatevaccine, Her2/new, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL theratope, BLP25(MUC-1), liposomal idiotypic vaccine, Melacine, peptide antigenvaccines, toxin/antigen vaccines, MVA-vased vaccine, PACIS, BCG vaccine,TA-HPV, TA-CIN, DISC-virus and ImmunCyst/TheraCys.

In a further aspect, the invention provides a method for treating cancerin a cancer patient comprising administering to the patient a monoclonalantibody in combination with an immunostimulatory oligonucleotide and/orimmunomer, as described herein. Passive immunotherapy in the form ofantibodies, and particularly monoclonal antibodies, has been the subjectof considerable research and development as anti-cancer agents. The term“monoclonal antibody” as used herein refers to an antibody molecule ofsingle molecular composition. A monoclonal antibody composition displaysa single binding specificity and affinity for a particular epitope.Accordingly, the term “human monoclonal antibody” refers to antibodiesdisplaying a single binding specificity which have variable and constantregions derived from human germline immunoglobulin sequences. Examplesof anti-cancer agents include, but are not limited to, Panorex(Glaxo-Welicome), Rituxan (IDEC/Genentech/Hoffman la Roche), Mylotarg(Wyeth), Campath (Millennium), Zevalin (IDEC and Schering AG), Bexxar(Corixa/GSK), Erbitux (Imclone/BMS), Avastin (Genentech) and Herceptin(Genentech/Hoffman la Roche). Antibodies may also be employed in activeimmunotherapy utilising anti-idiotype antibodies which appear to mimic(in an immunological sense) cancer antigens. Monoclonal antibodies canbe generated by methods known to those skilled in the art of recombinantDNA technology.

As used herein, the term “carrier” encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, or other materialwell known in the art for use in pharmaceutical formulations. It will beunderstood that the characteristics of the carrier, excipient, ordiluent will depend on the route of administration for a particularapplication. The preparation of pharmaceutically acceptable formulationscontaining these materials is described in, e.g., Remington'sPharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack PublishingCo., Easton, Pa., 1990.

The invention provides a kit comprising a chemotherapeutic agent, andimmunostimulatory oligonucleotides and/or immunomers, the lattercomprising at least two oligonucleotides linked together, such that theimmunomer has more than one accessible 5′ end, wherein at least one ofthe oligonucleotides is an immunostimulatory oligonucleotide. In anotheraspect, the kit comprises an immunostimulatory oligonucleotide and/orimmunostimulatory oligonucleotide conjugate and/or immunomer orimmunomer conjugate according to the invention, a chemotherapeutic agentand a physiologically acceptable carrier. The kit will generally alsoinclude a set of instructions for use.

The combination of immunomer structure with synthetic stimulatorymotifs, for example CpR(R=2′-deoxy-7-deazaguanosine), induced differentcytokine expression profiles than without synthetic stimulatory motifs.As a result, IMO compounds not only cause less adverse reactionsfrequently seen with whole bacterial products but also elicit morespecific immune responses depending on the type of cancer.

Repeated peritumoral application of IMO compounds resulted in stronginhibition or eradication of established syngeneic tumors CT26.CL25 andB16.F0. Peritoneal administration of IMO motifs also suppresseddisseminated B16.F0, CT26.WT or CT26.CL25 tumor growth in the peritonealcavity. Several immunological properties of IMO compounds account forthis therapeutic effect. Without wishing to be bound to any particulartheory, IMO motifs possibly induce rapid, acute phase reactions aroundtumor nodules, including recruitment and activation of macrophages,dendritic and NK cells and induction of cytokine secretion. Consistentwith activation of immune cells, serum IL-12 and circulating NK andmacrophage cells markedly increased within 4 hr and persisted for 24 hrfollowing IMO administration (data not shown). Such elevated cellularimmune responses could create a hostile environment for tumor cells.Further, the destruction of tumor cells in such environment providestumor antigens to nearby dendritic cells (DCs) and macrophages. IMOcompounds are shown to directly and rapidly promote antigen presentationby DCs and functional maturation of macrophages increasing surfaceexpression of MHC and costimulatory molecules. The activated antigenpresenting cells then lead to a strong adaptive T lymphocyte-mediatedspecific immune response in tumor bearing mice.

Besides the innate immunity, the treatment of mice bearing CT26 colontumor resulted in the development of strong adaptive immune responses.First, the IMO treatment of mice bearing tumors expressing β-gal as amodel antigen showed strong MHC class I restricted specific T cellresponses. Second, tumor bearing mice treated with IMO compounds werespecifically protected against subsequent challenge with the same tumorcells, suggesting the involvement of memory T lymphocytes. Thirdly,naïve mice adoptively transferred with splenic cells obtained from tumorbearing mice treated with IMO compounds developed specific antitumorimmunity and rejected the same tumor challenge.

Th2-type cytokines down-regulate antitumor immunity, and the activationof Th1 cell responses can enhance antitumor immunity. Therefore, a shiftto Th1-type cytokine production could be a plausible approach forimmunotherapy of cancers as well as treating viral infections. Highlevels of Th-2 cytokine, IL-4, are found in cultures of spleen cellsobtained from either PBS or non-CpG DNA control treated CT26.CL25 tumorbearing mice. In contrast, splenocytes from IMO treated mice bearing thesame tumor produced higher IFN-γ, indicating IMO compounds can reverseTh2-type cytokine production to Th1 responses in tumor bearing mice.Furthermore, IMO therapy induced a 5-fold increase (OD units) in thelevels of circulating β-gal specific IgG2a, resulting in a significantincrease in IgG2a/IgG1 ratio. Additionally, IL-12 ko mice failed torespond to IMO treatment suggesting a role for this Th-1 cytokine in IMOantitumor activity. Taken together, this clearly indicates that IMOcompounds strongly activate Th1 immune responses in tumor bearing mice.

Major limitations for chemotherapy alone or as a follow-up treatmentafter surgery are toxicity and drug resistance. Immunotherapy whencombined with surgery and chemotherapy may have advantages to clear theresidual tumor cells and reduce the drug dose. This is especially truefor IMO-based immunotherapy as it activated both innate and adaptiveimmune systems. IMO treatment may overcome the immune suppressiveeffects of chemotherapeutic agents as evidenced by significant increasein CD69+ and CD86+ cells in IMO 2-docetaxel combination treated tumorbearing mice compared with tumor bearing mice treated with docetaxelonly. Effects of conventional chemotherapy using docetaxel ordoxorubicin in B16.F0 melanoma or 4T1 breast carcinoma bearing micerespectively were markedly enhanced when combined with IMO compounds.

Taken together, the current results suggest that IMO compounds inducedstrong immunopharmacological and antitumor effects in vivo. Tumorexperiments in knockout mice suggest that Th1 cytokine, IL-12, isrequired for IMO induced antitumor effects. Moreover, the treatment withIMO compounds not only resulted in tumor regression, but also led to thedevelopment of strong tumor specific adaptive immune responses.Additionally, human specific IMO compounds show potent antitumoractivity in syngeneic tumor models. A synergistic effect was found withthe combination of chemotherapy agents and IMO treatment. Moreover, IMOcompounds showed immune cell activation following chemotherapy,suggesting combination therapy as a means for overcoming immunesuppression induced by chemotherapy. No IMO treatment-related toxicitywas observed in mice in any tumor model at the doses studied.

The examples below are intended to further illustrate certain preferredembodiments of the invention, and are not intended to limit the scope ofthe invention.

EXAMPLES Example 1 Synthesis of Oligonucleotides ContainingImmunomodulatory Moieties

Oligonucleotides were synthesized on a 1 μmol scale using an automatedDNA synthesizer (Expedite 8909; PerSeptive Biosystems, Framingham,Mass.), following the linear synthesis or parallel synthesis proceduresoutlined in FIGS. 5 and 6.

Deoxyribonucleoside phosphoramidites were obtained from AppliedBiosystems (Foster City, Calif.). 1′,2′-dideoxyribose phosphoramidite,propyl-1-phosphoramidite, 2-deoxyuridine phosphoramidite,1,3-bis-[5-(4,4′-dimethoxytrityl)pentylamidyl]-2-propanolphosphoramidite and methyl phosponamidite were obtained from GlenResearch (Sterling, Va.). β-L-2′-deoxyribonucleoside phosphoramidite,α-2′-deoxyribonucleoside phosphoramidite, mono-DMT-glycerolphosphoramidite and di-DMT-glycerol phosphoramidite were obtained fromChemGenes (Ashland, Mass.). (4-Aminobutyl)-1,3-propanediolphosphoramidite was obtained from Clontech (Palo Alto, Calif.).Arabinocytidine phosphoramidite, arabinoguanosine, arabinothymidine andarabinouridine were obtained from Reliable Pharmaceutical (St. Louis,Mo.). Arabinoguanosine phosphoramidite, arabinothymidine phosphoramiditeand arabinouridine phosphoramidite were synthesized at Hybridon, Inc.(Cambridge, Mass.) (Noronha et al. (2000) Biochem., 39:7050-7062).

All nucleoside phosphoramidites were characterized by ³¹P and ¹H NMRspectra. Modified nucleosides were incorporated at specific sites usingnormal coupling cycles. After synthesis, oligonucleotides weredeprotected using concentrated ammonium hydroxide and purified byreverse phase HPLC, followed by dialysis. Purified oligonucleotides assodium salt form were lyophilized prior to use. Purity was tested by CGEand MALDI-TOF MS.

Example 2 Analysis of Spleen Cell Proliferation

In vitro analysis of splenocyte proliferation was carried out usingstandard procedures as described previously (see, e.g., Zhao et al.,Biochem Pharma 51:173-182 (1996)). The results are shown in FIG. 8A.These results demonstrate that at the higher concentrations, Immunomer6, having two accessible 5′ ends results in greater splenocyteproliferation than does Immunomer 5, having no accessible 5′ end orOligonucleotide 4, with a single accessible 5′ end. Immunomer 6 alsocauses greater splenocyte proliferation than the LPS positive control.

Example 3 In Vivo Splenomegaly Assays

To test the applicability of the in vitro results to an in vivo model,selected oligonucleotides were administered to mice and the degree ofsplenomegaly was measured as an indicator of the level ofimmunostimulatory activity. A single dose of 5 mg/kg was administered toBALB/c mice (female, 4-6 weeks old, Harlan Sprague Dawley Inc, Baltic,Conn.) intraperitoneally. The mice were sacrificed 72 hours afteroligonucleotide administration, and spleens were harvested and weighed.The results are shown in FIG. 8B. These results demonstrate thatImmunomer 6, having two accessible 5′ ends, has a far greaterimmunostimulatory effect than do Oligonucleotide 4 or Immunomer 5.

Example 4 Cytokine Analysis

The secretion of IL-12 and IL-6 in vertebrate cells, preferably BALB/cmouse spleen cells or human PBMC, was measured by sandwich ELISA. Therequired reagents including cytokine antibodies and cytokine standardswere purchased form PharMingen, San Diego, Calif. ELISA plates (Costar)were incubated with appropriate antibodies at 5 μg/mL in PBSN buffer(PBS/0.05% sodium azide, pH 9.6) overnight at 4° C. and then blockedwith PBS/1% BSA at 37° C. for 30 minutes. Cell culture supernatants andcytokine standards were appropriately diluted with PBS/10% FBS, added tothe plates in triplicate, and incubated at 25° C. for 2 hours. Plateswere overlaid with 1 μg/mL appropriate biotinylated antibody andincubated at 25° C. for 1.5 hours. The plates were then washedextensively with PBS-T Buffer (PBS/0.05% Tween 20) and further incubatedat 25° C. for 1.5 hours after adding streptavidin conjugated peroxidase(Sigma, St. Louis, Mo.). The plates were developed with Sure Blue™(Kirkegaard and Perry) chromogenic reagent and the reaction wasterminated by adding Stop Solution (Kirkegaard and Perry). The colorchange was measured on a Ceres 900 HDI Spectrophotometer (Bio-TekInstruments). The results are shown in Table 5A below.

Human peripheral blood mononuclear cells (PBMCs) were isolated fromperipheral blood of healthy volunteers by Ficoll-Paque density gradientcentrifugation (Histopaque-1077, Sigma, St. Louis, Mo.). Briefly,heparinized blood was layered onto the Histopaque-1077 (equal volume) ina conical centrifuge and centrifuged at 400×g for 30 minutes at roomtemperature. The buffy coat, containing the mononuclear cells, wasremoved carefully and washed twice with isotonic phosphate bufferedsaline (PBS) by centrifugation at 250×g for 10 minutes. The resultingcell pellet was then resuspended in RPMI 1640 medium containingL-glutamine (MediaTech, Inc., Herndon, Va.) and supplemented with 10%heat inactivated FCS and penicillin-streptomycin (100 U/ml). Cells werecultured in 24 well plates for different time periods at 1×10⁶cells/ml/well in the presence or absence of oligonucleotides. At the endof the incubation period, supernatants were harvested and stored frozenat −70° C. until assayed for various cytokines including IL-6 (BDPharmingen, San Diego, Calif.), IL-10 (BD Pharmingen), IL-12 (BioSourceInternational, Camarillo, Calif.), IFN-α (BioSource International) and-γ (BD Pharmingen) and TNF-α (BD Pharmingen) by sandwich ELISA. Theresults are shown in Table 5 below.

In all instances, the levels of IL-12 and IL-6 in the cell culturesupernatants were calculated from the standard curve constructed underthe same experimental conditions for IL-12 and IL-6, respectively. Thelevels of IL-10, IFN-gamma and TNF-α in the cell culture supernatantswere calculated from the standard curve constructed under the sameexperimental conditions for IL-10, IFN-gamma and TNF-α, respectively.

TABLE 5Immunomer Structure and Immunostimulatory Activity in Human PBMC CulturesOligo Sequences and Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) No.Modification (5′-3′) or Each Chain D1 D2 D1 D2 255′-CTATCTGTCGTTCTCTGT-3′ 18mer (PS) 184 332 3077 5369 26

11mer (PS) 237 352 3724 4892 Oligo Sequences and Oligo Length/IL-10 (pg/mL) IFN-γ (pg/mL) No. Modification (5′-3′) or Each Chain D1 D2D1 D2 25 5′-CTATCTGTCGTTCTCTGT-3′ 18mer (PS) 37 88 125 84 26 11mer (PS)48 139 251 40 Oligo Sequences and Oligo Length/ TNF-α (pg/mL) No.Modification (5′-3′) or Each Chain D1 D2 25 5′-CTATCTGTCGTTCTCTGT-3′18mer (PS) 537 nt 26

11mer (PS) 681 nt D1 and D2 are donors 1 and 2.

TABLE 5AImmunomer Structure and Immunostimulatory Activity in BALB/c Mouse SpleenCell Cultures Oligo Sequences and  Oligo Length/ IL-12 (pg/mL)IL-6 (pg/mL) No. Modification (5′-3′) Or Each Chain 3 μg/mL 10 μg/mL 26

11mer (PS) 870 10670 27

11mer (PS) 1441 7664 28

11mer (PS) 1208 1021 29

11mer (PS) 162 1013 30

14mer (PO) 264 251 31

14mer (PO) 149 119 32

11mer (PS) 2520 9699 33

11mer (PS) 2214 16881 34

11mer PS) 3945 10766 35

11mer (PS) 2573 19411 36

14mer (PO) 2699 408 37

14mer (PO) 839 85 38

14mer (PO) 143 160Normal phase represents a phosphorothioate linkage; Italic phaserepresents a phosphodiester linkage.

In addition, the results shown in FIGS. 7A-C demonstrate that Immunomer2, with two accessible 5′ ends elevates IL-12 and IL-6, but not IL-10 atlower concentrations than Oligonucleotides 1 or 3, with one or zeroaccessible 5′ ends, respectively.

Example 5 Immunostimulatory Activity of Immunomers Containing aNon-Natural Pyrimidine or Non-Natural Purine Nucleoside

As shown in Tables 9-11, immunostimulatory activity was maintained forimmunomers of various lengths having a non-natural pyrimidine nucleosideor non-natural purine nucleoside in the immunostimulatory dinucleotidemotif.

TABLE 9 Immunomer Structure and Immunostimulatory Activity Oligo Length/IL-12 (pg/mL) @ IL-6 (pg/mL) @ No. Sequences and Modification (5′-3′) orEach Chain 3 μg/mL 3 μg/mL 51 5′-CTCACTTTCGTTCTCTGT-3′ 18mer 404 348 57

11mer 591 365 58

11mer 303 283 59

 8mer 55 66 60

 8mer 242 143

TABLE 10 Immunomer Structure and Immunostimulatory Activity IL-6 Oligo(pg/ Length/ IL-12 mL) or Each (pg/mL) 3 μg/ No. Sequences andModification (5′-3′) Chain 3 μg/mL mL 25 5′-CTATCTGTCGTTCTCTGT-3′ 18mer379 339 61

11mer 1127 470 62

11mer 787 296 63

 8mer 64 126 64

 8mer 246 113

TABLE 11 Immunomer Structure and Immunostimulatory Activity OligoLength/ IL-12 (pg/mL) IL-6 (pg/mL) No. Sequences and Modification(5′-3′) or Each Chain 3 μg/mL 3 μg/mL 4 5′-CTATCTGACGTTCTCTGT-3′ 18mer1176 1892 65

18mer 443 192 66

18mer 627 464 67

14mer 548 152 68

14mer 1052 1020 69

11mer 2050 2724 70

11mer 1780 1741 71

 8mer 189 55 72

 8mer 397 212

Example 6 Effect of the Linker on Immunostimulatory Activity

In order to examine the effect of the length of the linker connectingthe two oligonucleotides, immunomers that contained the sameoligonucleotides, but different linkers were synthesized and tested forimmunostimulatory activity. The results shown in Table 12 suggest thatlinker length plays a role in the immunostimulatory activity ofimmunomers. The best immunostimulatory effect was achieved with C3- toC6-alkyl linkers or abasic linkers having interspersed phosphatecharges.

TABLE 12 Immunomer Structure and Immunostimulatory Activity OligoLength/ IL-12 (pg/mL) IL-6 (pg/mL) No. Sequences and Modification(5′-3′) or Each Chain 0.3 μg/mL 1 μg/mL 4 5′-CTATCTGACGTTCTCTGT-3′ 18mer257 635 73

10mer 697 1454 74

10mer 1162 669 75

10mer 1074 1375 76

10mer 563 705 77

10mer 264 543 78

10mer 1750 2258 79

10mer 2255 2034 80

10mer 1493 1197 81

10mer 3625 2642 82

10mer 4248 2988 83

10mer 1241 1964

Example 7 Effect of Oligonucleotide Backbone on ImmunostimulatoryActivity

In general, immunostimulatory oligonucleotides that contain naturalphosphodiester backbones are less immunostimulatory than are the samelength oligonucleotides with a phosphorothioate backbones. This lowerdegree of immunostimulatory activity could be due in part to the rapiddegradation of phosphodiester oligonucleotides under experimentalconditions. Degradation of oligonucleotides is primarily the result of3′-exonucleases, which digest the oligonucleotides from the 3′ end. Theimmunomers of this example do not contain a free 3′ end. Thus,immunomers with phosphodiester backbones should have a longer half lifeunder experimental conditions than the corresponding monomericoligonucleotides, and should therefore exhibit improvedimmunostimulatory activity. The results presented in Table 13demonstrate this effect, with Immunomers 84 and 85 exhibitingimmunostimulatory activity as determined by cytokine induction in BALB/cmouse spleen cell cultures.

TABLE 13 Immunomer Structure and Immunostimulatory Activity OligoLength/ IL-12 (pg/mL) IL-6 (pg/mL) No. Sequences and Modification(5′-3′) or Each Chain 0.3 μg/mL 1 μg/mL 4 5′-CTATCTGACGTTCTCTGT-3′ 18mer225 1462 84

14mer 1551 159 85

14mer 466 467 L = C3-Linker

Example 8 In Vivo Anti-Cancer Activity of Immunomers in Combination withChemotherapeutic Agents

PC3 cells were cultured in 90% Ham's, F12K Medium with 10% Fetal BovineSerum (FBS), in presence of 100 U/ml Penicillin and 100 μg/mlStreptomycin to establish the Human Prostate cancer model (PC3). Maleathymic nude mice, 4-6 weeks old (Frederick Cancer Research andDevelopment Center, Frederick, Md.), were accommodated for 6 days forenvironmental adjustment prior to the study. Cultured PC3 cells wereharvested from the monolayer cultures, washed twice with Ham's, F12KMedium (10% FBS), resuspended in FBS-free Ham's, F12K Medium: Matrigelbasement membrane matrix (Becton Dickinson Labware, Bedford, Mass.)(5:1; V/V), and injected subcutaneously (5×10⁶ cells, total volume 0.2ml) into the left inguinal area of each of the mice. The animals weremonitored by general clinical observation, body weight, and tumorgrowth. Tumor growth was monitored by the measurement, with calipers, oftwo perpendicular diameters of the implant. Tumor mass (weight in grams)was calculated by the formula, ½a×b², where ‘a’ is the long diameter(cm) and ‘b’ is the short diameter (cm). When the mean tumor sizesreached ˜80 mg, the animals bearing human cancer xenografts wererandomly divided into the treatment and control groups (5animals/group). The control group received sterile physiological saline(0.9% NaCl) only. Immunomer 255 or 285, aseptically dissolved inphysiological saline, was administered by subcutaneously injection atdose of 0.5 or 1.0 mg/kg/day, 3 doses/week. Gemcitabine HCl (Eli Lillyand Company, Indianapolis, Ind.) was given twice by intraperitonealinjection at 160 mg/kg on Day 0 and 3. The detailed treatment scheduleis shown as follows.

-   -   G1: Saline    -   G2: Gemcitabine (160 mg/kg/day, IP, Day 0 and 3)    -   G3: 255 (1.0 mg/kg/day, SC, 3 doses/week, for 6 weeks)    -   G4: 255 (0.5 mg/kg/day, SC, 3 doses/week, for 6 weeks)    -   G5: 285 (1.0 mg/kg/day, SC, 3 doses/week, for 6 weeks)    -   G6: 285 (0.5 mg/kg/day, SC, 3 doses/week, for 6 weeks)    -   G7: 255 (0.5 mg/kg/day, SC, 3 doses/week, for 6        weeks)+Gemcitabine (160 mg/kg/day, Day 0 and 3)    -   G8: 285 (0.5 mg/kg/day, SC, 3 doses/week, for 6        weeks)+Gemcitabine (160 mg/kg/day, Day 0 and 3)

The tumor measurements after various treatments are presented in Table14 and FIG. 13. The tumor growth in all Immunomer 255 and 285 treatedanimals was remarkably inhibited compared with saline control (p<0.5).There was a tendency of dose-response relationship in these treatmentgroups (FIG. 13). There was no significant difference between Immunomers255 and 285 (Table 14).

TABLE 14 Tumor mass of tumor-bearing mice following treatment of 255,285, Gemcitabine or combination therapy Gemcitabine 255 255 160 1 0.5Day Saline SD SE mg/kg SD SE mg/kg SD SE mg/kg SD SE 0 82.7 16.7 7.582.6 15.7 7.0 80.1 10.6 4.7 80.4 10.5 4.7 3 81.9 13.3 5.9 73.0 3.4 1.567.5 8.1 3.6 54.3 8.4 3.7 6 80.5 11.5 5.2 50.4 11.7 5.2 50.4 9.0 4.045.3 5.5 2.5 9 87.7 8.2 3.7 35.7 6.3 2.8 40.9 5.1 2.3 43.9 9.3 4.2 1297.6 18.6 8.3 36.2 3.3 1.5 41.3 6.2 2.8 46.5 3.8 1.7 15 112.0 21.5 9.631.7 4.1 1.8 42.8 12.8 5.7 50.0 14.1 6.3 18 126.3 17.3 7.7 40.8 8.4 3.754.9 7.6 3.4 59.3 6.7 3.0 21 152.5 25.5 11.4 47.4 9.8 4.4 62.5 10.4 4.671.0 16.7 7.5 24 187.0 29.2 13.1 56.5 5.2 2.3 79.5 24.1 10.8 100.1 9.74.3 27 245.2 24.1 10.8 68.0 14.8 6.6 94.1 28.9 12.9 124.5 21.1 9.5 30343.6 63.9 28.6 89.4 11.1 5.0 119.8 18.7 8.3 162.4 37.5 16.8 33 438.5107.1 47.9 106.5 14.1 6.3 176.6 43.8 19.6 213.6 66.7 29.8 36 614.4 185.182.8 144.2 48.2 21.6 248.7 47.0 21.0 325.3 106.2 47.5 39 866.8 237.4106.2 175.3 61.4 27.5 320.1 64.2 28.7 416.8 154.5 69.1 42 1136.9 205.992.1 269.1 78.8 35.2 417.8 78.7 35.2 546.9 139.1 62.2 45 383.8 146.465.5 550.8 134.2 60.0 667.6 284.9 127.4 48 538.6 260.1 116.3 736.0 197.388.2 852.8 399.3 178.6 255 + 285 + 285 285 GEM GEM 1 0.5 0.5/160 0.5/160Day mg/kg SD SE mg/kg SD SE mg/kg SD SE mg/kg SD SE 0 80.4 11.0 4.9 79.910.3 4.6 79.4 10.1 4.5 78.7 12.0 5.4 3 52.3 9.3 4.2 64.7 9.0 4.0 45.18.2 3.7 44.6 8.7 3.9 6 38.8 4.6 2.1 46.9 14.7 6.6 31.2 5.9 2.6 34.7 4.42.0 9 34.5 9.5 4.3 43.5 13.6 6.1 22.1 4.8 2.1 23.0 3.2 1.5 12 35.8 9.44.2 43.0 15.9 7.1 15.0 3.8 1.7 11.9 2.2 1.0 15 36.6 8.7 3.9 48.6 15.46.9 18.0 3.1 1.4 12.4 3.5 1.6 18 45.1 14.6 6.5 62.0 20.2 9.0 17.9 3.11.4 15.5 1.7 0.8 21 53.5 12.3 5.5 73.6 20.5 9.2 18.3 2.8 1.2 14.8 2.11.0 24 72.6 22.7 10.1 93.6 23.0 10.3 23.6 4.5 2.0 23.0 1.5 0.7 27 86.513.7 6.1 119.3 17.3 7.8 27.8 4.1 1.8 25.9 3.7 1.7 30 114.5 22.8 10.2157.1 49.0 21.9 33.6 5.0 2.2 36.9 6.5 2.9 33 161.4 44.1 19.7 218.1 81.236.3 43.8 10.9 4.9 47.7 16.1 7.2 36 198.3 43.5 19.4 313.2 104.6 46.850.3 13.6 6.1 46.4 16.4 7.3 39 249.8 77.9 34.9 420.2 199.4 89.2 67.329.4 13.2 59.4 28.7 12.9 42 366.5 110.5 49.4 527.5 219.0 98.0 77.2 28.012.5 82.1 29.1 13.0 45 490.2 122.2 54.7 620.3 258.1 115.4 104.9 57.925.9 110.7 46.3 20.7 48 683.4 144.6 64.7 759.1 223.0 99.7 128.2 77.734.7 133.4 62.6 28.0 51 177.9 109.6 49.0 177.3 68.0 30.4 54 233.1 143.564.2 224.0 79.8 35.7 57 297.7 190.7 85.3 289.7 121.9 54.5

The body weight measurements after treatments at various times arepresented in Table 15 and FIG. 14. There was no significant differencein body weight gains among Immunomer 255 or 285 alone compared withcontrols. Gemcitabine treated animals had body weight loss in the firstweek and recovered in a week afterwards. Combination with Immunomer 255or 285 did not change the side effect profiles of Gemcitabine. No otherclinical abnormality or death was observed in all the groups.

TABLE 15 Body weights of tumor-bearing mice following treatment of 255,or saline. Gemcitabine 255 255 160 1 0.5 Day Saline SD SE mg/kg SD SEmg/kg SD SE mg/kg SD SE 0 24.1 2.5 1.1 23.5 0.9 0.4 23.2 1.4 0.6 23.02.4 1.1 7 25.8 3.0 1.3 20.7 4.4 2.0 25.2 2.4 1.1 24.8 2.8 1.2 14 26.83.2 1.4 25.2 4.0 1.8 26.3 2.0 0.9 26.0 2.9 1.3 21 28.2 3.3 1.5 27.1 3.91.7 27.8 2.0 0.9 27.6 2.8 1.2 28 29.4 3.5 1.6 28.1 4.3 1.9 28.6 2.6 1.128.0 2.7 1.2 35 30.6 3.7 1.6 29.4 2.9 1.3 29.5 2.3 1.0 28.6 2.8 1.3 4231.1 3.7 1.7 30.3 3.0 1.4 30.2 2.3 1.0 29.4 3.9 1.7 255 + 285 + 285 285GEM GEM 1 0.5 0.5/160 0.5/160 Day mg/kg SD SE mg/kg SD SE mg/kg SD SEmg/kg SD SE 0 22.5 1.3 0.6 24.1 1.6 0.7 21.9 1.7 0.7 23.0 0.8 0.4 7 24.30.9 0.4 25.6 2.0 0.9 19.1 2.0 0.9 22.3 3.3 1.5 14 25.1 1.3 0.6 27.0 2.10.9 24.6 1.6 0.7 25.9 2.7 1.2 21 26.1 1.3 0.6 27.8 1.5 0.7 26.8 1.6 0.727.1 2.6 1.2 28 27.2 1.5 0.7 28.3 2.2 1.0 27.2 1.6 0.7 27.7 3.2 1.4 3528.0 1.4 0.6 29.1 2.3 1.0 27.7 2.1 1.0 28.0 2.4 1.1 42 28.9 1.5 0.7 29.82.2 1.0 28.4 2.8 1.2 28.1 3.4 1.5

In summary, Immunomers 255 and 285 significantly inhibited tumor growthin nude mice bearing human prostate cancer PC3 xenografts with nosignificant side effects. When Immunomer 255 or 285 was given incombination with Gemcitabine, each compound significantly increased thetherapeutic effect of Gemcitabine without changes in side effectprofiles. In addition, there was a tendency in dose dependent responseof Immunomer 255 or 285 treatment.

Example 9 In Vivo Anti-Cancer Activity of Immunomers in Combination withChemotherapeutic Agents

The experiment of Example 8 was repeated using taxotere instead ofGemcitabine. Taxotere was administered on days 0 and 7. Immunomer 165was administered 5 days per week. Immunomers 255 and 285 wereadministered on days 0, 2, 4, 7, 9 and 11. The results are shown inTable 16 below. These results clearly demonstrate synergy between theimmunomers and taxotere.

TABLE 16 In vivo anti-cancer activity of immunomers in combination withother chemotherapeutic agents Taxotere 165 255 Day Saline SD SE (15mg/kg) SD SE (20 mg/kg) SD SE (1 mg/kg) SD SE 0.00 56.93 7.92 3.54 56.647.94 3.55 57.93 5.56 2.49 56.74 7.79 3.48 3.00 196.42 22.48 10.05 128.5120.83 9.32 95.79 16.04 7.18 87.12 6.64 2.97 6.00 708.85 32.64 14.60320.63 136.80 61.18 285.71 68.70 30.72 250.36 52.58 23.51 9.00 1370.95239.99 107.33 598.69 196.60 87.92 534.93 225.19 100.71 450.46 92.2541.26 12.00 2222.96 300.65 134.45 924.91 297.89 133.22 994.10 474.89212.38 814.21 197.16 88.17 15.00 3303.04 672.86 300.91 1589.08 578.38258.66 1601.73 576.19 257.68 1465.87 348.37 155.80 Taxotere + 255 285Taxotere + 165 SD SE (** mg/kg) SD SE (1 mg/kg) SD SE 55.51 9.55 4.2756.59 8.91 3.99 55.28 10.89 4.87 78.47 21.79 9.74 80.14 21.59 9.65 91.0123.60 10.55 211.52 88.59 39.62 216.85 89.40 39.98 303.00 61.33 27.43302.66 178.36 79.76 307.53 184.05 82.31 512.30 110.16 49.26 496.20342.69 153.25 510.18 351.16 157.04 884.12 308.22 137.84 686.47 385.97172.61 703.50 394.65 176.49 1479.21 416.64 186.33

Example 10

IMO compounds as shown in FIG. 15 and a non-CpG DNA:(5′-CTATCTCACCTTCTCTGT-3′) were synthesized, purified, and analyzed asdescribed above.

Female BALB/c (H-2^(d)), C57BL/6, and IL-6 and IL-12 knockout (ko) (bothko on a C57BL/6 background) mice 5-8 weeks of age, were purchased fromJackson Laboratory (Bar Harbor, Me.). CT26.WT (ATCC, Rockville, Md.) isa carcinogen-induced BALB/c undifferentiated colon carcinoma. CT26.CL25(ATCC, Rockville, Md.) is a subclone of CT26.WT that has been transducedwith Escherichia coli β-gal gene. 4T1 is a mammary adenocarcinoma cellline in BALB/c mice. B16.F0 is a C57BL/6 derived melanoma (ATCC).CT26.WT and 4T1 cells were cultured in RPMI 1640, 10% heat-inactivatedfetal bovine serum (FBS, Atlas Biologicals, Fort Colins, Colo.), 2 mML-glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin (Mediatech,Va.). CT26.CL25 is maintained in the same medium plus 400 μg/ml G418sulfate (Life Technologies, Grand Island, N.Y.). B16.F0 cells were grownin DMEM containing 10% FBS and antibiotics.

To assess serum cytokine levels, BALB/c mice (n=5) were injectedintraperitoneally (i.p), subcutaneously (s.c) or intramuscularly (i.m)with IMO compounds at 10 mg/kg (single dose). Sera was collected byretro-orbital bleeding at 4 hrs of IMO administration and determinedIL-12 and IL-6 by sandwich ELISA. Cytokine antibodies and standards werepurchased from PharMingen (San Diego, Calif.).

For the analysis of serum antibodies, 96 well plates were incubated atroom temperature for 3 hours with β-gal protein (Calbiochem Novabiochem,Pasadena, Calif.) at 2 μg/ml in phosphate buffered saline (PBS). Thesolid phase was incubated overnight at 4° C. with normal mouse serum(NMS) or antiserum, or β-gal specific monoclonal Ab (CalbiochemNovabiochem, Pasadena, Calif.) followed by an incubation withhorseradish peroxidase (HRP)-conjugated antibodies specific for mouseIgG (H+L). For isotype analysis, HRP-labelled goat anti-mouse IgG1 andIgG2a (Southern Biotechnology, Birmingham, Ala.) were used. The bindingof antibodies was measured as absorbance at 405 nm after reaction of theimmune complexes with ABTS substrate (Zymed, San Francisco, Calif.).

For subcutaneous solid tumor models, 10⁶ CT26.CL25 cells/mouse or 5×10⁵B16.F0 cells/mouse in 100 μl of PBS were implanted into BALB/c orC57BL/6 mice in the lower right flank. The tumor size reached to 50 to200 mg on day 6 for CT26.CL25 and on day 8 for B16.F0 post tumorinoculation. The tumor bearing mice were then treated with peritumoralinjection of IMO compounds or non-CpG DNA control at a dose of 1 mg/kgevery other day for 10 times. Tumor growth was recorded with the use ofcalipers, by measuring the long and short diameters of the tumor. Tumorvolumes were measured with a caliper and the formula (0.5×length×width²)was applied to determine tumor growth kinetics.

For peritoneal disseminated tumor model, 3×10⁵ CT26.WT or CT26.CL25cells and 5×10⁴ B16.F0 cells were injected i.p to BALB/c or C57BL/6 micerespectively. IMO compounds or non-CpG DNA (2.5 mg/kg) wereadministrated i.p. twice per week starting on day 1 for a total of 5times. Mice were checked daily for tumor growth and for survival. Eachdose group had 6 to 10 mice.

For the 4T1 tumor models, 5×10⁵ cells/mouse in 100 μl PBS were implantedinto BALB/c mice in the lower right flank. On day 5 when the averagetumor size reached 50 mm², the mice were given i.p injections of 30mg/kg doxorubicin (Bedford lab, Bedford, Ohio) for three times on days5, 6 and 7. IMO 2 (1 mg/kg) dissolved in 100 μl PBS was administrated byperitumoral injection at twice a week interval for a total of six times.

For B16.F0 melanoma tumor, C57BL/6 mice were injected i.p with 5×10⁴cells/mouse in 100 μl PBS on day 0. The mice were treated on day 2 withone i.p injection of 20 mg/kg docetaxel (Aventis, Bridgewater, N.J.) andthen given i.p injections of 2.5 mg/kg IMO 2 on days 3, 6, 9, 12 and 15.

Long term survivors (n=5) of IMO compound treated mice with CT26.WT orCT26.CL25 peritoneal tumor were rechallenged i.p. or i.v. with 5×10⁵ ofthe parental tumor cells without any further treatment. To evaluate thespecificity of the IMO induced antitumor response in tumor bearing mice,these long term survivors (n=5) were also rechallenged with syngeneic,non-organ-related mammary tumor 4T1 (5×10⁵). For i.v. rechallengedgroups, mice were sacrificed on day 13, lungs were harvested and lungmetastases were counted.

To study adoptive immune cell transfer, BALB/c mice were adoptively i.p.transferred with 5×10⁶ of syngeneic splenocytes either from naïve BALB/cmice or from IMO treated long term survivors bearing CT26.WT orCT26.CL25, the mice (5/group) were then cross challenged i.p. with 3×10⁵CT26.WT, CT26.CL25 or 4T1 cells on day 3.

To determine T cell responses, two or three mice from each group weresacrificed at day 26 after s.c. tumor implantation or day 21 after i.p.tumor inoculation, pooled T cells from splenocytes in each group werepurified using T cell enrichment columns (R&D systems, Minneapolis,Minn.). Purified T cells (2.5×10⁵) were stimulated with 2.5×10⁵mitomycin C— (50 μg/ml, Sigma, St. Louis, Mo.) treated β-gal or OVApeptide-pulsed syngeneic spleen cells for 24 hrs. T cells specificallyresponding to H-2^(d) restricted, antigen specific (β-gal₈₇₆₋₈₈₄)restimulation were then determined by interferon-gamma (IFN-γ) and IL-4ELISPOT analysis according to the manufacturer's directions (R&DSystems). Spots were enumerated electronically (Zellnet, New York,N.Y.).

Example 11 Serum Cytokine Secretion Profiles of IMO Compounds

IMOs 1 and 2 induce strong IL-12 secretion, while IMO 2 induced lowerIL-6 production in vitro (FIG. 16). To evaluate theimmunopharmacological effects in vivo, IMO compounds, CpG immunomers,and non-CpG oligos were administered to BALB/c mice i.p, s.c or i.m at adose of 10 mg/kg and their serum was evaluated for IL-12 and IL-6 after4 hrs. Both IMO compounds induced strong serum IL-12 secretion comparedwith a conventional CpG oligo (Table 17). IMO 2 containing a syntheticCpR motif, however, induced a significantly lower serum IL-6 in allthree routes of administration (Table 17) further confirming our earlierin vitro studies. The control non-CpG DNA showed insignificant IL-12 andIL-6 induction.

TABLE 17 In vivo cytokine induction^(a) by IMO compounds administered bydifferent routes. Intraperitoneal (i.p) Intramuscular (i.m) Subcutaneous(s.c) Oligo IL-12 IL-6 IL-12 IL-6 IL-12 IL-6 CpG DNA 36.0 ± 0.5 1.1 ±0.2 62.7 ± 6.4  0.6 ± 0.07 48.6 ± 6.9  0.3 ± 0.03 IMO 1 59.0 ± 11  5.9 ±0.2 109.3 ± 25  5.8 ± 0.5 98.3 ± 15  4.3 ± 0.3 IMO 2 51.7 ± 0.9 2.5 ±0.2 87.9 ± 3.2 1.2 ± 0.1 136.9 ± 17  2.3 ± 0.4 Non-CpG 0.86 ± 0.5 0.7 ±0.3  1.6 ± 0.07 Nd  1.7 ± 0.04 Nd ^(a)The values shown are averages inng/mL ± SD of three individual mouse; nd—not detectable.

Example 12 IMO Compounds Show Potent Antitumor Activity in Murine ColonCarcinoma Model

The antitumor activity of IMO compounds in a murine colon carcinomaCT26.CL25 model was evaluated. BALB/c mice bearing CT26.CL25subcutaneous solid tumors were treated with 1 mg/kg IMO compounds byperitumoral administration every other day for 10 times starting on day6 following tumor inoculation. Treatment with IMO compounds resulted incomplete rejection or strong inhibition of tumor growth in up to 75% ofanimals (FIG. 17A). An average tumor growth inhibition of 72% and 85%was observed in mice treated with IMOs 1 and 2, respectively, on day 24compared with non-CpG DNA treated mice. Furthermore, peritonealadministration of IMO 2 at a dose of 1 mg/kg to mice bearing peritonealdisseminated ascites CT26.WT (FIG. 17B) and CT26.CL25 (FIG. 17C)resulted in a marked increase of mice survival.

Example 13 Levels of Circulating β-Gal-Specific IgG1 and IgG2aSubclasses

The serum of CT26.CL25 tumor bearing mice following treatment with IMOcompounds for β-gal-specific IgG1 and IgG2a antibody levels wasanalyzed. The mice treated with IMO compounds showed over 5-foldincrease (OD units) in anti-β-gal-specific IgG2a levels (FIG. 18). Thetreatment with a conventional CpG DNA resulted in only about a 2-foldincrease in β-gal-specific IgG2a levels. In contrast, only a moderate0.5 to 2-fold increase in β-gal-specific IgG1 levels was observed (FIG.18).

Example 14 IMO Compounds Induce Tumor Specific CTL Responses

To examine if IMO treatment of tumor bearing mice resulted in tumorspecific CTL responses, T cells purified from splenocytes obtained frommice bearing CT26.CL25 tumor in different treatment groups werestimulated with mitomycin C-treated (3-gal or OVA peptide-pulsedsyngeneic spleen cells for 24 hrs. A significantly higher tumor specificCTL response to H-2^(d) restricted (β-gal₈₇₆₋₈₈₄) antigen was found inmice treated with IMO compounds and CpG DNA than in mice treated with acontrol non-CpG DNA as determined by higher IFN-γ induction (FIG. 19A),but not IL-4 (FIG. 19B).

Example 15 Persistent Antitumor Memory Following IMO Treatment

To study whether the IMO treatment would also induce tumor-specificadaptive immune response, mice that had been cleared of CT26.CL25peritoneal tumor by IMO treatment were rechallenged. Mice previouslytreated with IMO 2 rejected i.p rechallenge with CT26.WT and CT26.CL25tumors (FIGS. 20A and B). The mice that survived from peritonealinjected tumor after IMO 2 treatment were also able to reject pulmonarymetastases of the same tumor after i.v. inoculation (data not shown).Similar results were found in CT26.WT tumor model experimentsrechallenged with CT26.WT or CT26.CL25 cells (data not shown). Thesedata indicate that the mice treated with IMO 2 developed adaptive immuneresponse not only against model tumor antigen β-gal, but also againstparent tumor (CT26) antigens. However, such an immune memory was tumorspecific and the same mice were not protected from syngeneic,non-organ-related 4T1 mammary carcinoma challenge (FIG. 20C).

Example 16 Naïve Mice Develop Antitumor Protection Following AdoptiveTransfer of Immune Cells from IMO Treated Mice

Consistent with the concept that IMO 2 treatment induced specificantitumor immunity, splenocytes from the mice that rejected CT26.CL25tumor after IMO treatment were transferred to naïve mice, and these micewere challenged with CT26.CL25 or 4T1 tumor cells. Splenocytes from IMO2 treated mice, but not from naïve mice, were protective against alethal tumor challenge with CT26.CL25 tumor cells (FIG. 21A). As in thecase of tumor rechallenge experiment, this protection was tumor specificand did not extend to 4T1 tumor cell challenge (FIG. 21B).

Example 17 IMO 2 Shows Potent Antitumor Activity and Induces IgG2aAntibody Production in Murine Melanoma Model

IMO 2 was further tested for its antitumor activity in mice bearingB16.F0 murine melanoma. C57BL/6 mice bearing B16.F0 melanoma weretreated with 1 mg/kg IMO 2 by peritumoral administration every other dayfor 10 times starting on day 8 following tumor inoculation. As shown inFIG. 22A, IMO 2 caused a tumor growth inhibition of 71% in C57BL/6 micebearing subcutaneous B16.F0 melanoma. IMO 1 treatment also resulted insimilar levels of tumor inhibition as that of IMO 2 (data not shown).

As in the case of CT26.CL25 colon carcinoma, treatment of mice bearingB16.F0 tumor with IMO 2 resulted in a significant increase in totalcirculating serum IgG2a with a decrease or no change in total IgG1levels compared with control non-CpG DNA treated mice (FIGS. 22B and C).These results suggest the potent Th1 type immune responses in micebearing B16.F0 melanoma following IMO treatment.

Example 18 IMO Induced Th1 Type Responses are Essential for AntitumorProtection in Mice Bearing B16.F0 Melanoma

The data shown above including increases in IgG2a levels and tumorantigen specific CTL responses indicated a clear shift towards aTh1-dominated responses following IMO treatment in colon carcinomamodel. The antitumor effects of IMO compounds in wild-type (wt), IL-12ko, and IL-6 ko C57BL/6 mice bearing B16 melanoma were examined.Treatment of wt and IL-6 ko C57BL/6 mice bearing B16.F0 tumor with IMO 2resulted in a significant reduction in tumor growth (FIG. 23). However,IMO treatment had an insignificant effect on IL-12 ko mice bearing thesame tumor, suggesting that IL-12 is required for IMO induced antitumoractivity (FIG. 23).

Example 19 Synergy of Combination Treatment of ConventionalChemotherapeutic Agents and IMO Compounds

Synergistic effects between chemotherapeutic agents and IMO compounds inmice bearing B16.F0 ascites tumors or 4T1 subcutaneous solid tumors wereexamined. Peritumoral injection of IMO 2 and systemic administration ofdoxorubicin alone gave strong inhibition of 4T1 tumor growth (FIG. 24A).In combination, the two treatments were even more potent (FIG. 24A). Thecombination treatment of docetaxel and IMO 2 also showed significantsynergy against peritoneal disseminated B16.F0 melanoma resulting inenhanced survival of mice over those treated with either agent alone(FIG. 24B).

The effects of docetaxel and IMO 2 treatment on immune cell activationwere tested by determining the population changes of CD69+ and CD86+cells in peripheral blood. Mice treated with IMO 2 showed significantincrease in the percentage of CD69+ and CD86+ cells, while docetaxel at30 mg/kg given on days 1 and 3 did not inhibit such activations (FIG.24C).

Example 20 IMO 3 Containing Human-Specific Motif Shows Potent AntitumorActivity and Induces Th-1 Cytokine, IL-12, in Tumor Bearing Mice

Based on the results of IMO 2 that contained a mouse-specificimmunostimulatory motif, IMO 3, which contained a human-specific motif,was synthesized and studied its activity against CT26.CL25 tumor inmice. IMO 3 also showed potent antitumor activity against this tumormodel (FIG. 25A). As expected, both IMOs 2 and 3 induced IL-12 secretionin mice (FIG. 25B). Additionally, as shown in FIG. 26, activation ofhuman PBMCs by IMO 3 induces lysis of Her-2 positive BT-474 cells in thepresence of Herceptin.

Example 21 Enhanced Anti-Tumor Effect of Rituxan in Combination with IMOCompounds

Namalwa B-cell lymphoma cells were implanted in NOD/SCID mice throughintraperitoneal injection to generate a disease similar to humanhigh-grade B-cell Non-Hodgkins Lymphoma. Tumor bearing mice were treatedby intraperitoneal injections of 50 mg/kg Rituxan on days 4, 6 and 8and/or 2.5 mg/kg IMO 2 on days 4, 6, 8, 11, 14 and 21 (FIG. 27). Asshown in FIG. 28, tumor growth was significantly inhibited with thecombination of Rituxan and IMO 2.

Example 22 IMO Compounds Potentiate Anti-Tumor Effect of Herceptin

Nude mice bearing subcutaneously implanted Her-2 overexpressing humanbreast tumors (BT474) were treated by intraperitoneal injection of 10mg/kg Herceptin and/or peritumoral injection of 1 mg/kg IMO compoundtwice a week for 6 weeks (FIG. 27). Tumor growth after treatment withHerceptin or IMO 2 alone was inhibited 70% and 65% compared to the PBScontrol group (FIG. 29). A marked 97% suppression of tumor growth wasfound with combination treatment of Herceptin and IMO 2 (FIG. 29).

Example 23 IMO Compounds Potentiate Anti-Tumor Effect of Herceptin

Nude mice bearing subcutaneously implanted Her-2 overexpressing humanbreast tumors (BT474). Tumor bearing mice were treated byintraperitoneal injections of Rituxan on days 5, 7, 9 and 11 and/or IMO2 on days 5, 7, 9, 11 and 13. As shown in FIG. 30, tumor growth wassignificantly inhibited with IMO 2 and the combination of Rituxan andIMO 2.

EQUIVALENTS

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

1. A method for treating cancer in a cancer patient comprising administering to the patient an immunomer in combination with a chemotherapeutic agent, wherein the immunomer comprises at least one immunostimulatory oligonucleotide having the structure 5′-Nn-N1-Y—Z—N1-Nn-3′  (III) wherein: YZ is an immunostimulatory dinucleotide and Y is cytidine, 2′ deoxycytidine, arabinocytidine, 2′-deoxythymidine, 2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine, 2′-deoxy-4-thiouridine, other non-natural pyrimidine nucleosides, or 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; Z is guanosine or 2′-deoxyguanosine, 2′ deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2′ substituted-arabinoguanosine, 2′-O-substituted-arabinoguanosine, 2′ deoxyinosine or other non-natural purine nucleoside, provided however, that when Y is cytidine or 2′ deoxycytidine then Z is not guanosine or 2′ deoxyguanosine, and when Z is guanosine then Y is not cytidine or 2′ deoxycytidine, N1, at each occurrence, is a naturally occurring or a synthetic nucleoside or an immunostimulatory moiety selected from the group consisting of abasic nucleosides, arabinonucleosides, 2′-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, and nucleosides linked by a phosphodiester or modified internucleoside linkage to the adjacent nucleoside on the 3′ side, the modified internucleotide linkage being selected from, without limitation, a linker having a length of from about 2 angstroms to about 200 angstroms, C2-C18 alkyl linker, poly(ethylene glycol) linker, 2-aminobutyl-1,3-propanediol linker, glyceryl linker, 2′-5′ internucleoside linkage, and phosphorothioate, phosphorodithioate, or methylphosphonate internucleoside linkage; Nn, at each occurrence, is a naturally occurring nucleoside or synthetic nucleoside or an immunostimulatory moiety selected from the group consisting of abasic nucleosides, arabinonucleosides, 2′-deoxyuridine, α-deoxyribonucleosides, 2′-O-substituted ribonucleosides, and nucleosides linked by a modified internucleoside linkage to the adjacent nucleoside on the 3′ side, the modified internucleoside linkage preferably being selected from the group consisting of amino linker, C2-C18 alkyl linker, poly(ethylene glycol) linker, 2-aminobutyl-1,3-propanediol linker, glyceryl linker, 2′-5′ internucleoside linkage, and methylphosphonate internucleoside linkage; wherein each n is independently a number from 0 to 30; and wherein the 3′ end is linked directly or via a non-nucleotidic linker to another oligonucleotide, which may or may not be immunostimulatory. wherein the immunomer comprises at least two linked oligonucleotides and has more than one 5′ end, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide having an accessible 5′ end and comprises an immunostimulatory dinucleotide selected from the group consisting of C*pG, CpG* and C*pG*; wherein C is cytidine or 2′ deoxycytidine, wherein C* is selected from arabinocytidine, 2′-deoxythymidine, 2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine, 2′-deoxy-4-thiouridine, other non-natural pyrimidine nucleosides, or 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; wherein G is guanosine or 2′ deoxyguanosine, wherein G* is selected from 2′ deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2′ substituted-arabinoguanosine, 2′-O-substituted-arabinoguanosine, 2′ deoxyinosine or other non-natural purine nucleoside; wherein the chemotherapeutic agent is selected from the group consisting of gemcitabine, taxotere, doxorubicin, docetaxel, herceptin and rituxan; wherein the combination of the immunomer and chemotherapeutic agent create an enhanced anti-cancer effect relative to the immunomer or the chemotherapeutic agent alone.
 2. The method according to claim 1 wherein the purine is 6-thioguanine or 7-deazaguanine.
 3. The method according to claim 1 wherein the non-naturally occurring pyrimidine base is selected from the group consisting of 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, and 4-thiouracil.
 4. The method according to claim 1, wherein the non-nucleotidic linker selected from the group consisting of a linker from about 2 angstroms to about 200 angstroms in length, a metal, a soluble or insoluble biodegradable polymer bead, an organic moiety having functional groups that permit attachment to the 3′-terminal nucleoside of the oligonucleotide, a biomolecule, a cyclic or acyclic small molecule, an aliphatic or aromatic hydrocarbon, either of which optionally can include, either in the linear chain connecting the oligonucleotides or appended to it, one or more functional groups selected from the group consisting of hydroxy, amino, thiol, thioether, ether, amide, thioamide, ester, urea, and thiourea; amino acids, carbohydrates, cyclodextrins, adamantane, cholesterol, haptens, antibiotics, glycerol and a glycerol homolog of the formula HO—(CH₂)_(o)—CH(OH)—(CH₂)_(p)—OH, wherein o and p independently are integers from 1 to about 6, and a derivative of 1,3-diamino-2-hydroxypropane.
 5. The method of claim 1 further comprising administering a vaccine.
 6. The method of claim 5 wherein the immunomer or the vaccine, or both, are linked to an immunogenic protein.
 7. The method of claim 5 further comprising administering an adjuvant.
 8. The method according to claim 1, wherein at least one N1 or Nn is an immunostimulatory moiety.
 9. A pharmaceutical formulation comprising an immunomer, a chemotherapeutic agent and a physiologically acceptable carrier; wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotidic linker and having more than one 5′ end, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide having an accessible 5′ end wherein at least one oligonucleotide of the immunomer has the structure 5′-Nn-N1-Y—Z—N1-Nn-3′  (III) wherein: YZ is an immunostimulatory dinucleotide and Y is cytidine, 2′ deoxycytidine, arabinocytidine, 2′-deoxythymidine, 2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine, 2′-deoxy-4-thiouridine, other non-natural pyrimidine nucleosides, or 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; Z is guanosine or 2′-deoxyguanosine, 2′ deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2′ substituted-arabinoguanosine, 2′-O-substituted-arabinoguanosine, 2′ deoxyinosine or other non-natural purine nucleoside, provided, however that when Y is cytidine or 2′ deoxycytidine, then Z is not guanosine or 2′ deoxyguanosine, N1, at each occurrence, is a naturally occurring or a synthetic nucleoside or an immunostimulatory moiety selected from the group consisting of abasic nucleosides, arabinonucleosides, 2′-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, and nucleosides linked by a phosphodiester or modified internucleoside linkage to the adjacent nucleoside on the 3′ side, the modified internucleotide linkage being selected from, without limitation, a linker having a length of from about 2 angstroms to about 200 angstroms, C2-C18 alkyl linker, poly(ethylene glycol) linker, 2-aminobutyl-1,3-propanediol linker, glyceryl linker, 2′-5′ internucleoside linkage, and phosphorothioate, phosphorodithioate, or methylphosphonate internucleoside linkage; Nn, at each occurrence, is a naturally occurring nucleoside or synthetic nucleoside or an immunostimulatory moiety selected from the group consisting of abasic nucleosides, arabinonucleosides, 2′-deoxyuridine, α-deoxyribonucleosides, 2′-O-substituted ribonucleosides, and nucleosides linked by a modified internucleoside linkage to the adjacent nucleoside on the 3′ side, the modified internucleoside linkage preferably being selected from the group consisting of amino linker, C2-C18 alkyl linker, poly(ethylene glycol) linker, 2-aminobutyl-1,3-propanediol linker, glyceryl linker, 2′-5′ internucleoside linkage, and methylphosphonate internucleoside linkage; wherein each n is independently a number from 0 to 30; and wherein, the 3′ end is linked directly or via a non-nucleotidic linker to another oligonucleotide, which may or may not be immunostimulatory; and wherein the chemotherapeutic agent is selected from the group consisting of gemcitabine, taxotere, doxorubicin, docetaxel, herceptin and rituxan.
 10. The pharmaceutical formulation according to claim 9 wherein the purine is 6-thioguanine or 7-deazaguanine.
 11. The pharmaceutical formulation according to claim 9 wherein the non-naturally occurring pyrimidine base is selected from the group consisting of 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, and 4-thiouracil.
 12. The pharmaceutical formulation according to claim 9, wherein the non-nucleotidic linker selected from the group consisting of a linker from about 2 angstroms to about 200 angstroms in length, a metal, a soluble or insoluble biodegradable polymer bead, an organic moiety having functional groups that permit attachment to the 3′-terminal nucleoside of the oligonucleotide, a biomolecule, a cyclic or acyclic small molecule, an aliphatic or aromatic hydrocarbon, either of which optionally can include, either in the linear chain connecting the oligonucleotides or appended to it, one or more functional groups selected from the group consisting of hydroxy, amino, thiol, thioether, ether, amide, thioamide, ester, urea, and thiourea; amino acids, carbohydrates, cyclodextrins, adamantane, cholesterol, haptens, antibiotics, glycerol and a glycerol homolog of the formula HO—(CH₂)_(o)—CH(OH)—(CH₂)_(p)—OH, wherein o and p independently are integers from 1 to about 6, and a derivative of 1,3-diamino-2-hydroxypropane.
 13. The pharmaceutical formulation of claim 9 further comprising a vaccine.
 14. The pharmaceutical formulation of claim 13 wherein the immunomer or the vaccine, or both, are linked to an immunogenic protein.
 15. The pharmaceutical formulation of claim 13 further comprising an adjuvant.
 16. The pharmaceutical formulation according to claim 9, wherein at least one N1 or Nn is an immunostimulatory moiety. 